Compositions of influenza hemagglutinin with heterologous epitopes and/or altered maturation cleavage sites and methods of use thereof

ABSTRACT

Modified forms of hemagglutinin (HA) protein including those with modified immunodominant regions and with modified maturation cleavage sites, and virus and virus-like particles containing them are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase of PCT applicationPCT/US2017/016251 having an international filing date of 2 Feb. 2017,which claims benefit of U.S. provisional application No. 62/290,894filed 3 Feb. 2016. The contents of the above patent applications areincorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:758252000100_SubSeqList.txt, date recorded: Jan. 24, 2019, size: 185,944bytes).

TECHNICAL FIELD

This invention is in the field of immunology and virology. Moreparticularly it relates to compositions of recombinant influenzahemagglutinin (HA) proteins with altered and heterologous epitopesand/or altered maturation cleavage sites.¹ ¹ The following abbreviationsare applicable. HA, hemagglutinin; NA, neuraminidase; NAbs, neutralizingantibodies; bNAbs, broadly neutralizing antibodies; TEV, tobacco etchvirus; DNA, deoxyribonucleic acid; cDNA, complementary DNA; RNA,ribonucleic acid; kb, kilobase; kDa, kilo Dalton; CHO cells, Chinesehamster ovary cells; HEK 293 cells, human embryonic kidney 293 cells;VLP, virus-like particle; BEVS, baculovirus expression vector system;AcNPV, Autographa californica nuclear polyhedrosis virus; BmNPV, Bombyxmori nuclear polyhedrosis virus; TIPS, titerless infected-cellspreservation and scale-up; BIIC, baculovirus infected insect cells; PCR,polymerase chain reaction; FBS, fetal bovine serum; PDB, Protein DataBank which is on the World Wide Web at (rcsb.org/pdb/home/home.do); MOI,multiplicity of infection; MCS, maturation cleavage site; HPAI, highpathogenic avian influenza; HI, HA inhibition; WT, wild type; TIV,trivalent inactivated influenza vaccines; PBS, phosphate bufferedsaline.

BACKGROUND ART

Influenza, commonly known as “the flu”, is an infectious respiratorydisease caused by infection of human or zoonotic influenza viruses.Outbreaks of influenza occur annually during the cold months of eachyear, commonly known as “flu season”. These annual epidemics cause 3-5million cases of severe illness and up to 500,000 deaths worldwide,although most symptoms of influenza infections are mild (World HealthOrganization: Influenza (Seasonal) on the World Wide Web atwho.int/mediacentre/factsheets/fs211/en/). Each century, there areseveral influenza pandemics when influenza viruses infect a largeproportion of human population and inflict significant morbidity andmortality around the world. The 1918 Spanish flu pandemic that killed 3to 5 percent of the world's population is the most deadly pandemic inrecorded history.

Current Strategies to Elicit Stem Domain Reactive Antibodies

Elicitation of broadly neutralizing antibodies (bNAbs) against allinfluenza viruses has been the ultimate goal of flu vaccine design.Majority of the isolated bNAbs recognize conserved epitopes in the HAstem domain. However, antibodies elicited by natural influenza virusinfection or current flu vaccines mostly recognize the antigenic sitesthat surround the receptor binding site in the HA globular head domaindue to the immunodominance of these antigenic sites. These antibodiesare generally strain-specific as a result of the high variability ofthese antigenic sites among influenza virus strains. Thus current fluvaccines do not induce universal immunity against flu viruses.

Hemagglutinin (HA) is an integral membrane glycoprotein. It is the mostabundant protein in influenza virus lipid bilayer envelope. About 500molecules of HA are estimated on the surface of a virion. HA is the onlyprotein required for adsorption and penetration of virus into hostcells. It binds to host cell receptors and enables fusion between thevirion lipid envelope and the host cell membrane. Through its ability tobind host cell receptors, HA determines the host range of an influenzastrain.

HA is synthesized as a single chain precursor named HA0 in theendoplasmic reticulum (Stevens, J., et al., Science (2006) 312:404-410).The HA0 precursor has a signal peptide at its N-terminus and a membraneanchor sequence at its C-terminus. The N-terminal signal peptide isremoved during the process when HA is transported across and anchoredinto the host cell membrane. HA is assembled into trimers of identicalsubunits in the endoplasmic reticulum and is then exported to the cellsurface via the Golgi network. HA0 is cleaved at the C-terminal end ofthe maturation cleavage site (MCS) by specific host trypsin-likeprotease and converts to the mature form consisting of twodisulfide-bond linked polypeptides, HA1 and HA2. HA1 is the largerN-terminal portion and HA2 is the smaller C-terminal portion of HA0. HA2has the transmembrane region with a short C-terminal intracellular tailand anchors the HA to the membrane. Each mature HA has a total molecularweight of about 70 kDa with HA1 about 45 kDa and HA2 about 25 kDa. Theatomic structures of the extracellular portion of HAs from manyinfluenza strains have been determined. The structural model of a matureHA is shown in FIG. 1. All HAs, either as HA0 or the mature form, havethe same overall trimeric structure with a globular head on a stem. EachHA monomer is anchored on membrane by a single C-terminal transmembraneregion with a short hydrophilic cytoplasmic tail.

The globular head domain is made entirely of the HA1 and containsimmunodominant epitopes. The stem domain is mostly made of the HA2 andcontains conserved regions that are subdominant immunogenically. Theglobular head domain has an eight-stranded β-sheet structure at its corewith surface loops and helices. The membrane proximal stem domain iscomposed of left-handed superhelix of a triple coiled-coil structure ofresidues from both HA1 and HA2. Each HA monomer has multipleglycosylation sites with a total carbohydrate of about 13 kDa ofmolecular weight or 19% of the total HA molecular weight. Mostglycosylation is in the stem domain near the membrane surface. HA isalso modified by palmitoylation of cysteine residues on the cytoplasmictail (Veit, M., et al., J. Virol (1991) 65:2491-2500).

To circumvent the immunodominance of the globular head domain,immunogens have been designed based on the HA stem domain without theglobular head domain (headless HAs). These attempts have been largelyunsuccessful due to the difficulty of production of such molecules withproper tertiary structure (Krammer, F. and Palese, P. Curr Opin Virol(2013) 3:521-530; Eckert, D. M., and Kay, M. S., PNAS (2010)107:13563-13564). HA2 by itself has been expressed in E. coli in solubleform that folds into its most stable post-fusion low-pH-inducedconformation (Chen, J., et al., PNAS (1995) 92:12205-12209). Byincorporating designed mutations to destabilize the low pH conformationof HA2, another HA2 construct (HA6) based on H3 HA was expressed in E.coli and refolded into the desired neutral pH pre-fusion conformation(Bommakanti, G., et al., PNAS (2010) 107:13701-13706). HA6 was highlyimmunogenic in mice and protected mice against the infection byhomologous influenza A viruses. However, sera from HA6 immunized micefailed to neutralize virus in vitro, which could be due to limitation ofthe assay that only detect virus-neutralizing activity of the antibodiesrecognizing the globular head domain. Another “headless” HA stem domainconstruct with the HA2 and the region of HA1 in the stem domain has beendescribed (Steel, J., et al., mBio (2010) 1:e00018-10). This immunogenprotected mice against homologous influenza challenge and elicitedantisera cross-reactive to heterologous HAs from within the same group.As with HA6, the antisera did not show neutralizing activity in vitro.These designs are all based on protein minimization by eliminatingimmunodominant regions.

Recently, through structure-based rational design and reiterative bNAbselection of construct libraries, stable trimeric H1 HA stem-onlyimmunogens (headless mini-HAs) were made (Impagliazzo, A., et al.,Science (2015) 349:1301-1306; Yassing, H., M., et al., Nat Med (2015)21:1065-1070; Patent application WO2014/191435_A1). These stem-onlymini-HA immunogens elicited expected antibodies against HA stem domain.Mice and ferrets immunized with these mini-HA immunogens were protectedfrom lethal challenge of highly pathogenic H5 virus. In addition, amini-HA immunogen elicited H5 neutralizing antibodies in cynomolgusmonkeys. These mini-HA immunogens were selected for their binding tospecific bNAbs. The lengthy reiterative bNAb selection process needs tobe repeated for a different type of bNAbs. Although conformations of thebNAb epitopes were preserved in these mini-HA immunogens, the overallstructures were different from the stem domain of native HA. Due tothese structural changes, it is unlikely these headless mini-HAimmunogens can be assembled into influenza viruses or influenzavirus-like particles (VLPs). In addition, these headless mini-HAimmunogens do not have the receptor binding site for host cell binding.

Chimeric HAs have been designed to direct the host immune response tothe stem domain. Most neutralizing antibodies induced by pandemic H1N1infection were broadly cross-reactive against epitopes in the HA stemdomain and globular head domain of multiple influenza strains (Wrammert,J., et al., J. Exp Med (2011) 208:181-193). Immunization with HA derivedfrom H5N1 influenza strains (a group 1 HA) that is not circulating inhumans substantially increases HA stem-specific responses to group 1circulating seasonal strains (Ellebedy, A. H., et al., PNAS (2014)111:13133-13138; Nachbagauer, R., et al., J Virol (2014) 88:13260-13268;Whittle, J. R. R., et al., J. Virol (2014) 88:4047-4057). After eachemergence of pandemic influenza virus strains in 1957, 1968, and 2009,existing seasonal virus strains were replaced in the human population bythe novel pandemic strains.

It is hypothesized that exposure to new pandemic influenza virus strainswith divergent globular head domains lead to affinity matured memoryresponses to the conserved epitopes in the stem domains (Palese, P. andWang, T. T., mBio (2011) 2:e00150-11). In support of this hypothesis,engineered recombinant chimeric HA of H6, H9, or H5 globular head domainand H1 stem domain generated high titer stem-specific neutralizingantibodies (Pica, N., et al., PNAS (2012) 109:2573-2578; Krammer, F., etal., J. Virol (2013) 87:6542-6550). In these cases, the globular headdomain of H1 HA was replaced by the globular head domain of H6, H9, orH5 from the same group 1 HA to which most of the human population isnaïve. These replacements were made by replacing the H1 HA sequencebetween cysteine 52 and cysteine 277 of HA1 with the correspondingsequence of H6, H9, or H5. Cysteine 52 and cysteine 277 form a disulfidebond in the hinge region between the globular head domain and the stemdomain. Similar chimeric HAs were made with H3 stem domain to developvaccines for group 2 influenza strains (Krammer, F., et al., J. Virol(2014) 88:2340-2343; Margine, I., et al., J. Virol (2013)87:10435-10446). Immunizing animals with these chimeric HAs inducedstem-specific antibodies with broad neutralizing activity against eachgroup of viruses. Human population has preexisting immunity tocirculating H1 (group 1), H3 (group 2), and influenza B virus strains.Vaccination with a chimeric HA boosted antibody levels against the stemdomains that are common to HAs of the circulating strains and thechimeric HA. Only a primary response was induced against the novelglobular head domain on the chimeric HA to which humans are naïve.Subsequent boost with a second chimeric HA that possesses the same stemdomain but a different head domain further increased stem-specificantibody levels. The results suggest changing the host exposure to theimmunodominant epitopes in the globular head domain can increase thebroadly protective immune responses against the immunosubdominantepitopes in the stem domain.

The six-amino-acid loop of antigenic Site B of A/WSN/33(H1N1)hemagglutinin can be replaced by the homologous antigenic Site Bresidues of HAs from A/Japan/57(H2N2) and A/Hong Kong/8/68(H3N2) (Li,S., et al., J. Virol (1992) 66: 399-404). These replacements do notinterfere with the receptor binding function of HA. Recombinantinfluenza viruses with these chimeric HAs were replicated in MDCK(Madin-Darby Canine Kidney Epithelial Cells) cell culture. Viruses withchimeric HAs of A/WSN/33(H1N1) and A/Hong Kong/8/68(H3N2) inducedantibodies against both A/WSN/33(H1N1) and A/Hong Kong/8/68(H3N2). Theseresults suggest that the immunodominant antigenic sites of a HA can bereplaced by the homologous corresponding immunodominant antigenic sitesof other HAs from different strains. These replacements change theantigenic specificity of the resulting chimeric HAs.

In the foregoing paragraphs, therefore, the immunodominant epitopes inone influenza strain were replaced by the homologous immunodominantepitopes from another strain.

Another strategy is to dampen the immunodominant epitopes and refocusthe host immune response towards the stem domain. The immunodominantantigenic sites in the globular head domain can be shielded byintroducing additional glycosylation sites for hyperglycosylation(Eggink, D., et al., J. Virol (2014) 88:699-704, Patent applicationUS2014/0004149_A1). The hyperglycosylation in globular head domain didnot change the binding affinity of stem-reactive antibodies.Immunization of mice with the hyperglycosylated HA induced high titersof stem-reactive antibodies and protection against morbidity andmortality upon challenge with distinct seasonal viruses. Patentapplication US2013/0315929_A1 disclosed another method to dampen theimmunodominant epitopes in the globular head domain by replacing someresidues of those epitopes with other amino acids with less likelihoodof being part of an epitope. The results suggest shieldingimmunodominant epitopes in the globular head domain can increase thebroadly protective immune responses against the immunosubdominantepitopes in the stem domain.

HA as Carrier to Present Foreign Epitopes

Influenza HA has been used as a carrier for epitopes of V3-loop of HIV-1envelope protein. Insertion of immunodominant epitope peptides of 12 to22 residues in length from HIV-1 envelope protein gp120 to the HAimmunodominant antigenic site, either the Site A or Site B, producedchimeric HAs with individual HIV-1 epitope in the globular head domain.No residues of Site A and Site B were removed. The immunodominant HIV-1epitopes were inserted into the sites. The chimeric HAs induced immuneresponses to the HIV-1 V3-loop epitope in animals (Kalyan, N. K., etal., Vaccine (1994) 12:753-760; U.S. Pat. No. 5,591,823_A; Li, S., etal., J. Virol (1993) 67:6659-6666). The immunogenicity of the insertedepitopes appeared to be enhanced by HA since a very low dose of achimeric HA protein was sufficient to induce antibodies specific to theinserted epitope. These results suggest that immunodominant foreignepitopes from proteins other than HA can be inserted to theimmunodominant antigenic sites of HA. These chimeric HA molecules withinserted foreign immunodominant epitopes can induce immune responses tothe inserted foreign immunodominant epitopes.

DISCLOSURE OF THE INVENTION

This invention is directed to modified forms of influenza hemagglutininprotein, to vaccines, virus-like particles, and viruses that contain itas well as recombinant methods and materials for its production. Ingeneral, these modifications include replacement of the immunodominantregions of the globular head domain of HA protein with alternativeepitopes for generating antibodies and/or modification of the maturationcleavage site (MCS) in the stem domain of the HA protein. Cleavage ofHA0 between MCS and the fusion peptide to generate the free N-terminusof HA2 is essential for cell entry. By altering the MCS so as to preventits cleavage by animal proteases, a virus where the MCS has already beencleaved permits infection of host cells where the virus may multiply,but its progeny are uninfective. Thus, the presence of any epitopesincluding those occupying the immunodominant sites is amplified withoutfurther infection of the host by the progeny viruses.

In one aspect, the invention is directed to a flu vaccine whichcomprises a modified HA or a virus or virus-like particle which containsit, wherein an immunodominant region of the HA protein contains aconserved alternative epitope of the same influenza strain or anotherinfluenza strain inserted into this region. This provides a moresuccessful immunogenic form of the inserted alternative epitope that isimmunosubdominant in its native position. Alternative epitopes are thosenot derived from the globular head domain of other HAs. Typically theyare from the HA stem domains or from non-HA influenza proteins, such asM2.

In another aspect, the invention is directed to a modified influenzavirus (which could also be used as a vaccine) which has been modified tocontain an MCS that is not cleaved by animal proteases. As noted above,this permits amplification of the virus without engendering infectiveforms thereof. Such a virus can be further modified by replacing one ormore immunodominant regions with an alternative or heterologous epitopewhich may be an influenza epitope or a foreign epitope including, forexample, epitopes characteristic of other viruses, bacteria or tumorassociated antigens.

In still other aspects, the invention is directed to recombinantmaterials and methods for preparing the proteins, viruses or virus-likeparticles of the modified HA protein and methods to generate antibodiesusing these proteins, virus-like particles or modified viruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the X-ray crystal structure of the extracellularportion of a mature HA of A/California/07/2009, a swine origin influenzaA virus H1N1 strain (PDB:3LZG). This structure is used to guide theconstruct designs in this invention. The peptide sequence of this HA isused for making the parental wild type (WT) H1 HA construct. FIG. 1Ashows HA trimer with one HA monomer in ribbon drawing and the other twoin backbone drawing. The orientation with respect to the membrane, theposition of the globular head domain, and the stem domain are indicated.HA1 is shown as light shaded ribbon and HA2 is shown as dark shadedribbon. FIG. 1B shows the single HA monomer with the positions of HA1and HA2, as well the N- and C-termini of HA1 and HA2. The fusion peptideis circled.

FIG. 2 illustrates the approximate positions of the immunodominantantigenic sites of H3 HA mapped to the H1 HA monomer ofA/California/07/2009. FIG. 2A shows a ribbon drawing as in FIG. 1. TheH3 HA immunodominant antigenic sites A, B, C, D, and E are shown in darkshades. FIG. 2B is the top view of FIG. 2A above the globular headdomain distal to the membrane.

FIG. 3 illustrates the approximate positions of the immunodominantantigenic sites of H1 HA mapped to the H1 HA monomer ofA/California/07/2009. FIG. 3A shows a ribbon drawing as in FIG. 1. TheH1 HA immunodominant antigenic sites Ca1, Ca2, Cb, Sa, and Sb are shownin dark shades. FIG. 3B is the top view of FIG. 3A above the globularhead domain distal to the membrane. Many of these immunodominantantigenic sites surround the host cell receptor binding site asindicated by an arrow.

FIG. 4 illustrates the schematic design of the H1 HA constructs. FIG. 4Ais a schematic drawing of nucleic acid features of the constructs.Positions of restriction sites for subcloning are labeled. FIG. 4B is aschematic drawing of the protein features of the constructs in relativeproportion to the nucleic acid shown in FIG. 4A. GP67ss represents theGP67 secretion signal (signal peptide) at the N-terminus. HA1 representsHA HAL MCS represents HA maturation cleavage site which is part of HALHA2 represents HA HA2. TEV represents TEV cleavage site. Foldonrepresents foldon sequence. His represents 10-histidine-tag (10×His-tag)at the C-terminus.

FIG. 5 contains sequences H3 (SEQ ID NO: 63), WT (SEQ ID NO: 64) and H1(SEQ ID NO: 65) and illustrates the positions where the heterologousepitopes are placed in the HA globular head domain and where the alteredmaturation cleavage sites are. The parental construct peptide sequence(WT) is aligned with HA sequences of H3 (A/Aichi/2/1968 H3N2) and H1(A/Puerto Rico/8/1934 H1N1). The amino acid positions of each sequenceare labeled on the right based on HA0 numbering with the first residueafter signal sequence removal as position 1. The maturation cleavagesite (MCS) is indicated by an arrow and a vertical line. Theimmunodominant antigenic sites A, B, C, D and E of H3 HA are markedabove the H3 sequence. The immunodominant antigenic sites Ca1, Ca2, Cb,Sa and Sb of H1 HA are marked below the H1 sequence. The underlinedsequences in WT are replaced by the heterologous epitopes indicatedbelow the sequences. Altered maturation cleavage sites are indicatedbelow the maturation cleavage site (MCS). The native immunosubdominantantigenic sites for broadly neutralizing antibodies (bNAbs), FI6, 1C9,and CR8020, are marked by lines above the sequences, respectively.

FIG. 6 illustrates models of HA monomer with the composite CR8020epitope placed at the immunodominant antigenic sites of the globularhead domain. The HA monomer is shown in a ribbon drawing as in FIG. 1.The immunodominant antigenic sites that are replaced by composite CR8020epitope are shown in dark shade. The original native CR8020 epitopes inthe stem domain are also shown in dark shade. FIG. 6A shows the model ofCR8020Sa4. FIG. 6B shows the model of CR8020Ca.

FIG. 7 shows the expression data of HA constructs described in thisinvention. Cell culture medium of SF9 cells infected with recombinantbaculovirus of the indicated construct was harvested and captured byNi-NTA resin (Ni pull-down). The Ni-NTA resin was washed with 1×PBS toremove unbound proteins. Proteins bound to the Ni-NTA resin wereanalyzed by SDS-PAGE with Coomassie® staining (FIGS. 7A, B, C, and D) oranti-His Western blot (FIG. 7E). The arrow indicates the full-lengthHA0.

FIG. 8 shows the purified HA constructs used in this invention. Cellculture medium of SF9 cells infected with recombinant baculovirus of theindicated construct was harvested and captured by Ni-NTA resin (Nipull-down). The Ni-NTA resin was washed with 1×PBS. Bound proteins wereeluted with imidazole and analyzed by SDS-PAGE with Coomassie® staining.The arrow indicates the full-length HA0.

MODES OF CARRYING OUT THE INVENTION

As this invention is directed to modifications of HA protein, furtherdescription of this protein and its function in addition to thatprovided above may be helpful.

Protease cleavage of HA0 is a prerequisite for the infectivity ofinfluenza A viruses. The distribution of these proteases in the host isone of the determinants of tissue tropism and, as such, pathogenicity.Several trypsin-like proteases have been identified in respiratory trackand lung that cleave majority of HAs with monobasic maturation cleavagesites (Kido, H., et al., J. Biol Chem (1992) 267:13573-13579; Peitsch,C., et al., J. Virol (2014) 88:282-291; Zhirnov, O., P., et al., J.Virol (2002) 76: 8682-8689). These proteases are serine proteases. Oneof them is trypsin-like protease Clara, first isolated from ratbronchiolar epithelial Clara cells. Narrow tissue distribution of theseproteases that cleave HA0 restricts influenza virus infection to therespiratory tracks and lungs in mammals.

The proteases responsible for HA maturation are not yet wellcharacterized. Like trypsin, these proteases cleave the peptide bondC-terminal to a basic residue such as arginine (R) or lysine (K). Thematuration cleavage site (MCS) is located at the C-terminal end of HA1and cleavage at the C-terminus of MCS is essential for infectivity.

A polybasic sequence in the maturation cleavage sites of H5 and H7subtypes leads to cleavage susceptibility by a broader range of cellularproteases such as furin and subtilisin-type proteases, which correlateswith broader tissue tropism and higher pathogenicity of these viruses inmammals (Stieneke-Gröber, A., et al., EMBO J (1992) 11:2407-2414;Maines, T. R., et al., J. Virol (2005) 79:11788-11800). Many highlypathogenic avian influenza (HPAI) subtypes are H5 and H7. During HPAIoutbreaks in human, reported case-fatality rates are higher than thoseof pandemic and seasonal influenza viruses (Morens, D. M., et al.,Nature (2012) 486:335-340). These polybasic sequences in the MCS alsolead to viral replication in multiple organs of avian species, resultingin high mortality in these avian species.

The HAs of seasonal flu viruses and nonpathogenic avian influenzaviruses are cleaved extracellularly by specific proteases in respiratorytract and lung which limit their tissue tropism. On the other hand, theHAs of highly pathogenic viruses are cleaved intracellularly byubiquitously occurring proteases. These highly pathogenic virusesundergo multiple-cycle replication in various tissues to cause systemicinfections (Steinhauer, D. A., Virus. Virol (1999) 258:1-20;Taubenberger, J. K., PNAS (1998) 95:9713-9715). The 1918 pandemic strainof human influenza virus also utilizes a broad range of cellularproteases and use its neuraminidase (NA) to recruit plasminogen for HAcleavage (Goto, H. and Kawaoka, Y. PNAS (1998) 95:10224-10228; Chaipan,C., et al., J. Virol (2009) 83:3200-3211).

Cleavage of HA0 generates HA2 with a new free N-terminus which isessential for virion fusion with host cells. The cleaved form is themature form of HA with full function of receptor binding and membranefusion. HA0 has the ability to bind receptors but does not mediatemembrane fusion. Both the precursor and the mature form of HA exist onthe surface of virions. Viruses with only HA0 have no fusion activityand do not cause infection.

The N-terminal twelve residues of HA2 is called the “fusion peptide”(Skehel, et al., Biochem Soc Trans (2001) 29:623-626). The fusionpeptide has a hydrophobic sequence. In HA0, MCS and the fusion peptideform a surface loop. After cleavage, the newly generated N-terminalfusion peptide inserts into the HA trimer interface. Following bindingto host cell receptors, the attached influenza virions are endocytosedby the host cells into endosomes. The fusion potential of mature HA isactivated at endosomal pH, between pH 5 and 6 depending on theparticular influenza virus strain. Extensive changes in HA structure atlow pH result in extrusion of the fusion peptide towards the cellmembrane. Insertion of fusion peptide into the host endosomal membraneleads to fusion of the surrounding host endosomal membrane with theviral membrane containing the C-terminal membrane anchor region of HA.Fusion releases the viral RNA segments into the cytoplasm of the hostcell to enter into the host cell nucleus, where viral replicationoccurs.

HA determines the host range of an influenza virus strain. HA binds toterminal sialic acid of host cell surface glycoproteins and glycolipids.HAs from human influenza strains bind almost exclusively tosialyloligosaccharides terminated by SAα2,6Gal, whereas HA from avianand equine influenza strains bind SAα2,3Gal. Human has mostly SAα2,6Galsialyloligosaccharides and avian species have mostly SAα2,3Galsialyloligosaccharides. As schematically shown in FIG. 3, the hostreceptor binding sites on HA are located in the globular head domainthat is exclusively made of HA1. The receptor specificity of influenzavirus HA has been well characterized. The amino acid residuesresponsible for the recognition of SAα2,6Gal or SAα2,3Gal have beenmapped. It has been shown that a single amino acid residue substitutionin the receptor binding pocket changes the receptor binding specificity(Rogers, G. N., et al., Nature (1983) 304:76-78). Different isolates of1918 influenza pandemic virus had different receptor binding specificityas a result of a single amino acid substitution in the receptor bindingsite of HA (Glaser, L., et al., J. Virol (2005) 79:11533-11536). Thestrain A/South Carolina/1/18 HA preferentially binds the human cellularreceptor SAα2,6Gal, whereas the strain A/New York/1/18 HA binds bothhuman cellular receptor SAα2,6Gal and avian cellular receptor SAα2,3Gal,revealing the dynamic nature of host adaptation of influenza viruses.

Even though a single mutation in the receptor binding site of HA changesthe receptor binding specificity from avian to human, efficienttransmission of H5 avian influenza virus strain in human requiresseveral other changes in HA outside the receptor binding site andchanges in other influenza proteins (Imai, M., et al., Nature (2012)486:420-428; Herfst, S., et al., Science (2012) 336:1534-1541; Chen,L-M., et al., Virol (2012) 422:105-113; Russell, C. A., et al., Science(2012) 336:1541-1547). Together with other changes, as few as four aminoacid substitutions in H5 HA are sufficient to enable the transmission ofmutant avian H5 virus among ferrets through respiratory droplets. Inaddition to understanding of influenza virus transmission acrossspecies, these studies established laboratory procedures to studynon-mammalian influenza virus transmission in mammals, which can be usedto evaluate universal influenza vaccine candidates.

Description of Anti-Flu Antibodies and Antigenic Sites of HA

As the most abundant protein of influenza virus lipid bilayer envelope,HA is the major antigen of influenza virus and harbors the primaryepitopes for neutralizing antibodies. Most human anti-flu antibodies areagainst HA and NA. Sixty percent (60%) of the influenza-reactiveantibodies elicited by vaccination react to HA (Wrammert, J., et al.,Nature (2008) 453:667-671). Majority of HA-reacting antibodies recognizethe antigenic sites that surround the receptor binding site in theglobular head domain. Some of these antibodies are virus-neutralizingantibodies. These neutralizing antibodies (NAbs) generally interferewith HA binding to host cells and show HA inhibition (HI) activity. Theyare generally strain-specific due to the high variability of theseantigenic sites, and thus lack the much-desired broad neutralizingactivity (Wang, T. T., and Palese, P., Nat Struct Mol Biol (2009)16:233-234).

The widespread infection of influenza viruses is the result of theability of the virus to alter its antigenic properties. The changes ofantigenic properties of influenza viruses are the result of the lowfidelity replication of the viral genome by the error-prone viralRNA-dependent RNA polymerase complex. The high mutation rate leads toantigenic drift, a gradual change of antigenic properties of HA.Antigenic drift occurs in all types of influenza viruses. Sequenceanalyses of HA genes show that most of the changes in amino acidsequences are located in the HA1 even though silent nucleic acidsequence changes are spread over the entire HA gene (Palese, P., andYoung, J. F., Science (1982) 215:1468-1474). HA2 is more conserved thanHAL

The segmented nature of viral genome also leads to re-assortment ofviral genome segments when viruses of two different strains co-infect ahost at the same time. A new strain of virus emerges from thisre-assortment with different viral surface proteins that lead toantigenic shift, an abrupt change of antigenic properties of HA.Antigenic shift occurs only in Influenza A viruses. Antigenic drift andantigenic shift enable influenza viruses to escape from neutralizationby existing antibodies.

H3 Influenza

The first detailed structure of an influenza HA determined over 35 yearsago shed light on the antibody-binding sites of HA and presents amolecular explanation to antigenic drift and antigenic shift (Wilson, I.A., et al., Nature (1981) 289:366-373; Wiley, D. C., et al., Nature(1981) 289:373-378; Wiley, D. C., and Skehel, J. J., Ann Rev Biochem(1987) 6:365-394).

Comparison of HA sequences of antigenically distinct viruses identifiedimmunodominant antigenic sites in the globular head domain of H3 HA, agroup 2 subtype HA. The descriptions of immunodominant antigenic sitesof H3 HA are substantiated by others (Both, G. W., et al., J. Virol(1983) 48:52-60). The approximate positions of these antigenic sites ina HA structure are schematically shown in FIG. 2. The sequence positionsof these antigenic sites are shown in FIG. 5. Site A is located in thesurface loop of residues 133 to 148 of the HA of A/Aichi/2/1968 H3N2strain (numbering as H3 in FIG. 5). This loop, known as the 140-loop,projects from the globular head domain and is on the lower rim of thereceptor binding pocket. Site B is located on the top of the globularhead domain and comprises the surface α helix of residues 187 to 196 andadjacent surface loop of residues 155 to 160 along the upper rim of thereceptor binding pocket. Site C surrounds the disulfide bond betweenCys52 and Cys277. Crossing of the loop (residues 46 to 55) centered atCys52 and the loop (residues 271 to 280) centered at Cys277 forms abulge at the hinge between the globular head domain and the stem domain.Site D resides in the interface region between the HA monomer subunitsof an HA trimer. It centers at two β-strands of residues 200 to 214 inthe eight-stranded β-sheet structure in the core of the globular headdomain. Residues at the turns of the other β-strands may also be part ofSite D. Site D is mostly buried in the HA trimer interface. It is notclear how Site D functions as an antigenic site. Site E is locatedbetween Site A and Site C on the side of the globular head domain and ismade of surface loops of residues 62 to 63, residues 78 to 83, and theβ-strand of residues 119 to 122 on the edge of the eight-strandedβ-sheets. Together these residues of Site E form a continuous surface onthe side of the globular head domain. Comparing the amino acidsubstitutions and antigenic properties of influenza viruses that emergedduring the period from 1968 to 2003 showed that substitutionsresponsible for major antigenic changes were located exclusively in SiteA and Site B (Smith, D. J., et al., Science (2004) 305:371-376; Koel, B.F., et al., Science (2013) 342:976-979). Substitutions in Sites C, D andE appeared to cause minor antigenic changes. The results suggest thatmost strain-specific neutralizing antibodies bind to Site A and Site Bin the periphery of the receptor binding site of the globular headdomain.

H1 Influenza

Genetic analyses of antigenically distinct group 1 H1 HA of viral strainA/PR/8/34 identified distinct antigenic sites in the globular headdomain, termed Ca1, Ca2, Cb, Sa and Sb sites (Caton, A. J., et al., Cell(1982) 31:417-427; Gerhard, W., et al., Nature (1981) 290:713-717). Theapproximate positions of these antigenic sites in the HA structure areschematically shown in FIG. 3. The sequence positions of these antigenicsites are shown in FIG. 5. Ca1 site is located at the turns of theβ-strands of the eight-stranded β-sheet structure. One of the turns ofresidues 165 to 169 (numbering as H1 in FIG. 5) is surface exposed.Residue 207 of Ca1 site is at the turn connecting the two β-strandscorresponding to the Site D of H3 HA. Ca1 site of H1 HA generallycorresponds to Site D of H3 HA. Ca2 site is also made of two segmentsthat are separated in the primary structure but together in the tertiarystructure. One segment of Ca2 is in the surface loop of residues 136 to141 at the corresponding position of the Site A of H3 HA. Anothersegment of Ca2 is made of residues 220 to 221 on a long surface loop.Ca2 site is on the opposite side of Ca1 in the globular head domain of aHA monomer but is adjacent to Ca1 site of another HA monomer in a HAtrimer. Ca1 site of one HA monomer forms a continuous surface with theCa2 site of another HA monomer in the trimer structure. Cb site is alinear epitope of residues 70 to 75 that form a surface loop next to theeight-stranded β-sheet structure. It corresponds to Site E of H3 HA.Sites Sa and Sb can be considered as substitutes that correspond to SiteB of H3 HA. Site Sa of residues 154 to 163 overlaps with the loopcorresponding to Site B loop of H3 HA. Another segment of Sa site is anearby turn of residues 124 to 125. Site Sb corresponds to Site Bα-helix of H3 HA and mostly has the α-helix residues 188 to 194.

Broadly Neutralizing Antibodies and Conserved Epitopes

Natural influenza virus infection or vaccination with trivalentinactivated influenza vaccines (TIV) also elicit low levels ofantibodies against conserved HA epitopes that are mostly located in themembrane proximal stem domain (Ellebedy, A. H., et al., PNAS (2014)111:13133-13138). These antibodies recognize epitopes conserved amongmany strains of influenza viruses. Those that provide protection againstseveral strains are termed broadly neutralizing antibodies (bNAbs).These bNAbs generally do not have HA inhibition activity and do notprevent HA binding to host cell surface receptors. The firstcross-strain antibodies C179 has been characterized over 20 years ago(Okuno, Y., et al., J. Virol (1993) 67:2552-2558). It recognizes aconformational epitope of residues 318 to 322 (TGLRN) of HA1 andresidues 47 to 58 (GITNKVNSVIEK) of HA2 that are conserved among H1 andH2 strains. C179 inhibits the fusion activity of HA and thus results invirus neutralization.

Many bNAbs have been detected or isolated from patients infected withinfluenza viruses (Ekiert, D. C., and Wilson, I. A., Curr Opin Virol(2012) 2:134-141). The bNAbs have been demonstrated to neutralize group1, group 2, or both group 1 and group 2 influenza A viruses. Many of theantigenic epitopes recognized by bNAbs have been identified andcharacterized. These epitopes are either a linear segment of HA sequenceor conformational epitopes with multiple linear segments of HA sequence.Many of the epitopes to bNAbs are located in the less variable stemdomain of HA proteins. These epitopes include but are not limited to thefusion peptides and the peptide sequences around the MCS.

Many bNAbs and their epitopes have been structurally characterized. Forexample, bNAb FI6 recognizes residues of the MCS and the fusion peptidein HA0 (Corti, D., et al., Science (2011) 333:850-856). Peptide mappingof HA identified two peptides as FI6 epitopes, RKKRGLFGAIAGFIEconsisting of the MCS and most of the fusion peptide andKESTQKAIDGVTNKVNS of a helix coiled-coil peptide in HA2. The proposedneutralization mechanism of FI6 is to inhibit membrane fusion as well asto prevent HA maturation by blocking access of protease to the MCS ofHA0. Another bNAb F10 recognizes a conformational epitope around thefusion peptide in the mature form of HA (Su, J., et al., Nat Struct MolBiol (2009) 16:265-273). F10 inhibits all group 1 influenza A virusespresumably by preventing membrane fusion.

Monoclonal bNAb 1C9 inhibits cell fusion in vitro (U.S. Pat. No.8,540,995; Prabhu, N., et al., J. Virol (2009) 83:2553-2562). 1C9recognizes a linear epitope of GLFGAIAGF, the N-terminus of the fusionpeptide of the H5 HA2 of HPAI H5N1 (Immune Epitope Database web address:iedb.org/assay details.php?assayId=1599077). 1C9 shows protection inmice against infection by highly pathogenic avian influenza (HPAI) H5N1virus. Monoclonal bNAbs have also been isolated from memory B cells (Hu,W., et al., Virol (2013) 435:320-328). Several of these monoclonalantibodies recognize a linear epitope of FIEGGWTGMVDGWYGYHH of HA2 from2009 pandemic H1N1 influenza virus. This epitope is C-terminal to the1C9 epitope of the fusion peptide. The sequences of the 14 residues ofHA fusion peptides are highly conserved across influenza A and Bviruses.

The conserved nature of HA fusion peptides has been explored to developuniversal influenza vaccine. A peptide conjugate vaccine, based on thehighly conserved sequence of the HA0 of the influenza B virus thatincludes the last 9 amino acid residues of HA1 that contains the MCS andthe fusion peptide of HA2 at both sides of the scission bond, elicited aprotective immune response against lethal challenge with virus strainsof antigenically distinct lineages of influenza B viruses (Bianchi, E.,et al., J. Virol (2005) 79:7380-7388).

Monoclonal bNAb CR6261 recognizes a highly conserved region in HA2 helixA and HA1 residues in the stem domain (Ekiert, D. C., et al., Science(2009) 324:246-251). CR6261 neutralizes group 1 influenza viruses bypreventing conversion of HA to the post-fusion conformation. CR6261belongs to bNAbs that use the same V_(H)1-69 germline antibody heavychain. Another V_(H)1-69 monoclonal antibody, CR8020 neutralizes group 2influenza viruses. CR8020 binds HA at the base of the stem domain inclose proximity (˜15 to 20 Å) to the membrane, analogous to thoseantibodies against HIV that recognize the membrane-proximal externalregion (MPER) from the HIV gp41 subunit (Ekiert, D. C., et al., Science(2011) 333:843-850). The two main components of CR8020 epitope consistof the C-terminal portion (HA2 residues 15 to 19, EGMID of H3) of thefusion peptide and the outermost strand (HA2 residues 30 to 36, EGTGQAAof H3) of the 5-stranded β sheet near the base of the stem domain. Thesetwo components are 10 residues apart in the primary structure of H3.Most bNAbs that bind to the stem domain of HA neutralize either group 1(H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16) or group 2 (H3, H4, H7,H10, H14, and H15) of HAs of influenza A viruses. These antibodies donot inhibit HA binding to host cells but may prevent fusion of viralmembrane and host cell membrane.

The HA receptor binding site is a pocket at the top of the globular headdomain (Wilson, I. A., et al., Nature (1981) 289:366-373; Wiley, D. C.,and Skehel, J. J., Ann Rev Biochem (1987) 56:365-394). This pocket isformed by amino acid residues highly conserved across many influenzastrains. The rim of the pocket is formed by the immunodominant antigenicsites, such as Site A and Site B in H3 as described above. Cloning ofhuman monoclonal antibodies (mAbs) from healthy human subjectsidentified bNAbs that recognize conserved residues in close proximity tothe receptor binding site in the globular head domain of HA from H1, H2,and H3 strains (Krause, J. C., et al., J. Virol (2011) 85:10905-10908;Krause, J. C., et al., J. Virol (2012) 86:6334-6340). Structural studiesrevealed that at least some of these bNAbs mimic the interaction of thesialic acid to the receptor binding pocket (Whittle, J. R. R., et al.,PNAS (2011) 108:14216-14221; Ekiert, D. C., et al., Nature (2012)489:526-532).

Stem-reactive antibodies are rare in natural infection and even less inimmunization with current seasonal flu vaccines. Only a subset of theseantibodies are neutralizing antibodies. Due to the conserved nature ofthe stem domain, most of the neutralizing antibodies are bNAbs. Cloningfrom antibody libraries made from human subjects can now routinelyidentify these rare broadly neutralizing stem-reactive antibodies(Kashyap, A. K., et al., PNAS (2008) 105:5986-5991; Wrammert, J., etal., Nature (2008) 453:667-671). The rare occurrence of thesestem-reactive antibodies leads to the hypothesis that these epitopes inthe stem domain are immunosubdominant whereas the epitopes in theglobular head domain are immunodominant to which most antibodies aredirected (Krammer, F., and Palese, P., Nature Rev Drug Disc (2015)14:167-182). Repeated exposure to prevalent seasonal flu viruses orimmunization with seasonal flu vaccines leads to production ofantibodies against the immunodominant antigenic sites in the globularhead domain. It has been suggested that the presence of theimmunodominant epitopes in the globular head domain may skew secondaryresponses away from the stem domain (Russell, C. J., N Engl J Med (2011)365:1541-1542). Individuals who have been infected with current strainsor vaccinated against them may have a harder time mounting a universalresponse than those who are immunologically naïve.

M2 Protein and its Epitope

M2 is a single-pass transmembrane protein that forms a homotetramerproton channel in the viral envelope (Lamb. R. A., et al., Cell (1985)40:627-633; Pielak, R. M., and Chou, J. J., Biochim Biophys Acta. (2011)1808:522-529). It exists in much lower abundance compared to HA (1:10 to1:100 ratio of M2:HA) in the viral envelope. M2 proton channel functionis important to regulating the pH of viral interior for releasing viralproteins into the cytosol of host cells and to regulating the pH of theGolgi lumen for HA transport to the cell surface. Influenza A virus M2(AM2) protein has 97 residues with an extracellular N-terminal domain ofresidues 1 to 23 known as M2e, a transmembrane (TM) domain of residues24 to 46, and an intracellular C-terminal domain of residues 47 to 97.The TM domains from four M2 molecules form a four-helix bundle thatfunctions as a pH-sensitive proton channel to regulate pH across theviral membrane during cell entry and across the trans-Golgi membrane ofinfected cells during virus assembly and exit. The large cytoplasmicdomain is crucial for a stable tetramer formation and plays a role invirus assembly, through its association with the M1 protein of virioninner shell. Except for the HXXXW sequence motif in the TM domain thatis essential for proton channel function, the M2 proteins of influenzaA, B and C viruses share almost no sequence homology. However, the 10N-terminal extracellular residues of AM2 are conserved in all influenzaA viruses.

The influenza A virus M2 (AM2) is the target of the antiviral drugsamantadine and rimantadine which block the AM2 proton channel. The drugrimantadine, which stabilizes the closed state of the proton channel,binds to a lipid-facing pocket near the C-terminal end of the channeldomain. Amantadine is an antiviral drug with activity against influenzaA viruses, but not influenza B viruses. The use of these channelblockers has been discontinued due to widespread drug resistance as aresult of mutations in the channel domain of AM2. Many of thesemutations give rise to somewhat attenuated viruses that are lesstransmissible than wild type (WT) viruses. These drug resistant mutantscan revert to WT in the absence of drug selection pressure.

M2 protein is an integral membrane protein expressed abundantly on thesurface of host cells infected with influenza viruses (Lamb, R. A., etal., Cell (1985) 40:627-633). It is suggested that M2 is a cell surfaceantigen for cytotoxic T lymphocyte (CTL) response to influenza virus.Infection of influenza A viruses only elicit low titers of antibodiesagainst M2 (Feng, J., et al., Virol J. (2006) 3:102-115). The highdegree of structural conservation of M2e could in part be theconsequence of a poor M2e-specific antibody response and thus theabsence of pressure for change. Anti-M2 antibody responses were morerobust among individuals with preexisting antibodies to M2 protein(Zhong, W., et al., J. Infect Dis (2014) 209:986-994). The anti-M2antibodies induced as a result of infection with 2009 pandemic H1N1influenza A virus were cross-reactive to M2 protein of seasonalinfluenza A viruses. Treating mice with anti-M2e antibodiessignificantly delayed progression of the disease and led to isolation ofM2e escape mutants, suggesting the potential of using M2e as a vaccineagainst influenza A virus infection (Zharikova, D., et al., J. Virol(2005) 79:6644-6654).

A DNA vaccine based on the fusion of M2e with HA has been described(Park, K. S., et al., Vaccine (2011) 29:5481-5487). This fusion proteinhas one human M2e peptide and one avian M2e peptide positioned at theN-terminus of HA protein through a 20-residue linker between each other.The expression of the encoded M2e-HA fusion protein was confirmed. Miceimmunized with the M2e-HA fusion DNA vaccine showed enhanced T cellresponse to M2e and complete protection from lethal challenge ofheterologous avian influenza virus.

A recombinant fusion protein comprising of four tandem repeats of M2egenetically fused to the C-terminus of Mycobacterium tuberculosis HSP70(mHSP70) protein showed protection against multiple strains of influenzaviruses in mice (Ebrahimi, S. M., et al., Virology (2012) 430:63-72).The M2e peptide, SLLTEVETPIRNEWGCRCNDSSD, has been conjugated to acationic liposome delivery vehicle along with peptides from otherinfluenza proteins and the BM2, the homolog of M2e of Influenza B virusas vaccine (Patent Application US2010/086584_A1). The vaccine elicitedimmune response against M2e in mice. Mice vaccinated with the M2epeptide containing vaccine were protected from lethal influenza viruschallenge. An inactivated influenza vaccine supplemented with M2e VLPconferred improved and long-lasting cross protection againstantigenically different influenza A viruses in mice (Song, J-M., et al.,PNAS (2011) 108:757-761).

Modification of Immunodominant Regions

This invention includes genetically modified recombinant influenzahemagglutinin (HA) genes and proteins that result from replacing theimmunodominant regions of the globular head domain of HA withalternative epitopes or inserting the alternative epitopes therein todirect the host immune response to these epitopes. Some of theseepitopes are recognized by bNAbs to HAs. The invention is also directedto modified HA proteins and genes with modified MCSs and to combinationsof these modifications.

In one embodiment, modified recombinant HAs have the immunodominantantigenic sites of the globular head domain replaced by theextracellular domain of the M2 protein (M2e), or M2e is inserted intothe immunodominant antigenic sites.

Guided by the three dimensional structures of HA proteins, a specificsurface peptide or such surface peptides of immunodominant regions arereplaced by a heterologous peptide or several heterologous peptides ofother regions of the same HA or a heterologous peptide or severalheterologous peptides from a HA of another subtype or strain ofinfluenza virus. These heterologous peptides may be inserted into theimmunodominant regions. In addition, the specific surface peptide orpeptides of HA are replaced by heterologous peptides from other proteinsthat are not related to HA, or such peptides are inserted therein. Theseheterologous peptides are either natural proteins or artificiallydesigned. They may not be recognized by currently known antibodies.

The immunodominant regions of HA are on the surface of the globular headdomain of HA. These surface regions to be modified are surface-exposedhelices, β-strands, or loops. These surface regions are preferablyimmunodominant antigenic sites and epitopes or parts of such antigenicsites and epitopes or next to such antigenic sites and epitopes.

In some embodiments, the immunodominant regions of HA globular headdomain are replaced by peptides of the immunosubdominant epitopes of HAthat are recognized by bNAbs to HAs. In other embodiments, theimmunodominant regions of HA globular head domain are replaced bypeptides of the immunosubdominant epitopes that are recognized byantibodies that neutralize a group of influenza viruses. Theseimmunosubdominant epitopes are selected from the HA stem domain, orinclude the HA fusion peptide or the HA maturation cleavage site.

The recombinant modified HA proteins of this invention are expressed incell culture and secreted into the cell culture media at levels similarto recombinant wild-type HA. These designs are applicable to anyinfluenza HA of all strains of influenza A, B and C viruses, includingbut not limited to the influenza viruses that infect human, and theinfluenza viruses that infect other mammalian and avian species. In oneembodiment, these modified HA proteins can be used as immunogens to makevaccines against influenza virus infection in human or animals.

These immunodominant regions may be changed by site-directedmutagenesis. The solvent-exposed residues of an antigenic site areidentified by the three-dimensional structures of HA proteins. Asolvent-exposed residue or several solvent-exposed residues of anantigenic site are changed by site-directed mutagenesis or replaced witha peptide containing the specific changes of these residues. The newsite has the same secondary structure as the original site.

Illustrative HA with Antigenic Sites Replaced by Heterologous Epitopes

Based on the HA structure as illustrated in FIG. 3, several positions inHA are selected to illustrate the feasibility of replacingimmunodominant antigenic sites in the globular head domain byheterologous peptides from the stem domain of HA, for example.

Peptide KTSS of residues 119 to 122 (numbering as WT in FIG. 5) is asurface-exposed helical structure near the Sa site residues 124 to 125.The Sa site is on the side of the globular head domain of HA trimer andaway from the receptor binding site. Modification at this site isunlikely to change the receptor binding site and hence the host receptorbinding by the modified HA. The four-residue peptide KTSS is replaced bybNAb epitopes of the stem domain. HAGAKS of residues 137-142 at the Ca2site that corresponds to the Site A of H3 HA, is a major immunodominantsite. KKGNSYPKLSKS at residues 153 to 164 is a surface loop at the Sasite that corresponds to the Site B of H3 HA. The N-terminal part ofthis peptide KKGNS at residues 153 to 157 is replaced in someconstructs. TSADQQSLYQNA at residues 184 to 195 of Sb site forms thehelix at the top of the HA globular head domain.

The surface loop of the Sa site and helix of the Sb site are adjacent toeach other in the globular head domain of HA and correspond to the SiteB of H3. Together the Sa site and Sb site can accommodate aconformational epitope with loop and helix structures. However, sincethese sites are near the receptor binding site, replacement of thesepeptides may destroy the receptor binding site. EIAIRPKVRDQE at residues213 to 224 of Ca2 site is a loop near the interface between two HAmonomers. This loop has contacts with the β-strands of the adjacent HAmonomer that corresponds to the Site D of H3 HA. Part of the loop issurface-exposed. Ca2 site is located within this peptide. In someconstructs, this peptide is partially replaced.

In addition, the heterologous peptides can be placed in or near anyother antigenic sites as illustrated in FIG. 5, and surface loops orhelices as illustrated in FIG. 1 to FIG. 3. Furthermore, a heterologouspeptide can be inserted into any of these positions without any deletionof residues of the antigenic sites.

The bNAb CR8020 epitope is located in the stem domain. As shown in FIG.5 and FIG. 6, the CR8020 epitope has two main components consisting ofthe outermost β strand (HA2 residues 30 to 36, EGTGQAA of H3 HA, SEQ IDNO:26) of the 5-stranded β-sheet near the base of the stem domain andthe C-terminal portion (HA2 residues 15 to 19, EGMID of H3 HA, SEQ IDNO:25) of the fusion peptide. These two components are separated by 10amino acid residues in primary structure. A composite CR8020 epitopeEGMIDYEGTGQAA (SEQ ID NO:27) is designed in which the two epitopecomponents are linked by a tyrosine (Y). This composite CR8020 epitopeis used as a heterologous peptide to replace the immunodominant sites.

Another bNAb 1C9 epitope is the fusion peptide in the N-terminus of HA2present in the stem domain. A modified 1C9 epitope peptide ofGIFGAIAGFIEG (SEQ ID NO:36) was designed as a peptide to be displayed inan immunodominant site. This modified 1C9 peptide, denoted as I1C9, hasisoleucine (I) substitution of leucine (L) at the position 2 of the H5fusion peptide recognized by bNAb 1C9. This I1C9 fusion peptide ispresent in swine H1 HA and HAs of some H6 and H9 viruses. This I1C9peptide is placed at different immunodominant sites. Multiple constructshave been made with I1C9 to an antigenic site with shifted positions.

The bNAb FI6 epitope is a conformational epitope containing two peptidesthat form a continuous surface on the tertiary structure of HA precursorHA0 but separated in the primary structure (FIG. 5). One FI6 epitopepeptide RKKRGLFGAIAGFIE is the maturation cleavage site and fusionpeptide of HA0. Another FI6 peptide KESTQKAIDGVTNKVNS has a coiled-coilstructure in HA2. A construct has been made wherein these two FI6epitope peptides were placed in the globular head domain. The FI6epitope peptide RKKRGLFGAIAGFIE is placed at the Sa site of residues 153to 164 and the coiled-coil FI6 epitope peptide KESTQKAIDGVTNKVNS isplaced at the Sb site of residues 184 to 195. In another construct, theFI6 epitope peptide RKKRGLFGAIAGFIE is placed at the Sa site without thesecond FI6 epitope peptide.

Many bNAb epitopes have been characterized. Any of them can be placed ator near the immunodominant antigenic sites or surface loops in theglobular head domain as described above.

In other embodiments, the heterologous peptides are peptides of theextracellular domain of M2 proteins (M2e peptides). M2e peptides areconserved and less variable among influenza A viruses. M2e peptides arealso conserved and less variable among influenza B viruses, although theM2e peptides from the influenza A viruses and influenza B viruses aredifferent.

In some embodiments, the heterologous peptides are artificially designedpeptides that make specific changes to the immunodominant antigenicsites by site-directed mutagenesis. In some embodiments, an artificialpeptide combines desirable features of an immunodominant antigenic siteand a heterologous peptide. In some embodiments, an artificial peptidehas the residues for interaction with a bNAb and residues of theoriginal immunodominant antigenic sites to maintain the threedimensional structure of the HA globular head domain. These artificialpeptides are either rationally designed based on the HA threedimensional structures or by screening a library of randomly generatedpeptides.

Constructs have been made to place M2e peptide SLLTEVETPTRNGWECKCSDS ateither the Ca2 site or the Sb site for expression using baculovirusexpression system or mammalian expression system. The M2e peptide can befurther optimized based on consensus of sequences from differentinfluenza strains.

HA with Modified Maturation Cleavage Site

This invention includes embodiments wherein the protease susceptibilityof the HA maturation cleavage site (MCS) is changed to make theresulting HA susceptible to a different class of proteases and resistantto those trypsin-like proteases that cleave the native HA maturationcleavage sites of all strains of influenza viruses. An altered MCS isdesigned to be recognized by a specific protease that is not present inknown natural hosts of influenza viruses. This makes the resulting HAresistant to maturation in all natural hosts of influenza viruses. Inthe presence of this specific protease that recognizes this alteredmaturation cleavage site, these resulting HAs are cleaved to the matureform containing HA1 and HA2. Recombinant influenza viruses made from theresulting HAs in the presence of this specific protease form mature HAand become infectious to natural influenza hosts. However, the viralprogeny replicated in infected natural hosts are not infectious due tothe lack of the proper proteases in natural hosts.

H1 HA molecules often have a single basic residue in the MCS. Thissingle basic residue is usually an arginine (R) residue, N-terminal tothe scission bond. Adding multiple basic residues, such as arginine (R)or lysine (K), in the MCS of a H1 HA increases the infectivity of therecombinant influenza viruses containing the modified HA. Replacing anative H1 MCS with H5 MCS that contains polybasic residues increases theinfectivity of the recombinant influenza viruses containing the modifiedHA (Kong, W-p., et al., PNAS (2006) 103:15987-15991). These polybasicresidues are more susceptible to cleavage by many intracellulartrypsin-like proteases. In some embodiments, H1 HA MCS is modified byreplacing it with H5 HA MCS or polybasic residues. The H1 HA MCS is alsomodified by inserting polybasic residues between the last residue(arginine) of HA1 and the first residue (glycine) of HA2.

Further disclosed are genetically modified recombinant influenza HAgenes and proteins with altered MCS sequences. The native MCS isreplaced by sequences from the MCS from another HA. The said changesinclude replacing the native MCS of a HA with another MCS from adifferent HA that are known to make recombinant influenza viruses moreinfectious, presumably by making the resulting HA more susceptible tomaturation cleavage by unidentified host proteases. In the preferredembodiment, the native MCS of H1 HA is replaced by an MCS of H5 HA, orreplaced by polybasic residues of arginine (R) and lysine (K).

Several mammalian proteases such as Factor Xa and enterokinase havetheir respective cleavage recognition site located completely on theN-terminal side of the scission bond. Their cleavage recognition sitecan also be used to replace the native HA maturation cleavage site. Asin the case of the native HA, the C-terminal side of the scission bondof these protease cleavage sites is a glycine (G). The thrombin cleavagesite also has a glycine (G) on the C-terminal side of the scission bond.After thrombin cleavage, a new N-terminus of glycine (G) is generated.All these proteases are present in many natural influenza hosts. Howeverthese proteases are usually either not present or in trace amount incell culture media or cell lines used for recombinant HA production.

TEV protease of tobacco etch virus (TEV) recognizes a cleavage site ofENLYFQG and cleaves between glutamate (Q) and glycine (G). The freeN-terminus generated by TEV protease cleavage is glycine (G), the sameas N-terminal residue of HA2 after HA maturation. Unlike the MCS ofnative HA, there are no basic residues in TEV cleavage site. In someembodiments, the entire HA MCS is replaced by the TEV cleavage site, ora few residues of the MCS are replaced by the TEV cleavage site, or onlythe arginine (R) N-terminal to the scission bond, for example, isreplaced by the TEV cleavage site. The HA constructs with the MCSsreplaced by TEV cleavage site are expressed using baculovirus expressionsystem or mammalian expression system, and constructs that express tothe same level as wild-type HA have been identified.

In the presence of TEV protease that cleaves the modified MCS, the saidmodified HA undergoes maturation and is converted to HA1 and HA2 withthe native N-terminus of fusion peptide. The mature modified recombinantHA has the capability to bind the corresponding host cell receptors andundergo membrane fusion. In the absence of the TEV protease, the newlysynthesized said modified HA remains as the uncleaved HA0 precursor. Thelack of free N-terminus of fusion peptide prevents the said HA frommembrane fusion. The progeny HA protein produced in the infected hostcells remains as HA0 and is not processed to the mature functional formdue to the lack of TEV protease in the host. The newly made viruses withHA0 do not have the ability of membrane fusion and therefore arenon-infectious.

Combinations of Antigenic Sites and Maturation Cleavage Sites

The changes of the antigenic sites in the globular domain of HA and thechanges of MCS in the stem domain of HA can be in any kind ofcombinations. A particular heterologous peptide can be introduced to anantigenic site in the globular head domain of HA proteins with differentaltered maturation cleavage sites. In addition, two or more heterologouspeptides can be introduced to a single HA protein at different antigenicsites. In one embodiment, a genetically modified recombinant influenzaHA has M2e peptide replacing an antigenic site in the globular headdomain of HA and TEV protease site as the MCS. In another embodiment, agenetically modified recombinant influenza H1 HA has M2e peptidereplacing an antigenic site in the globular head domain of HA and H5 MCSin place of H1 native MCS.

Methods of Production

For production of modified HA, the protein is secreted by the cells intothe cell culture medium as soluble form and is not an integral membraneprotein or is not attached to cell membranes or cell surface. In someembodiments, the native HA signal sequence is used for secretion ininsect cells and in mammalian cells. In other embodiments, the native HAsignal sequence is replaced by an insect cell signal sequence forsecretion in insect cells or a mammalian signal sequence for secretionin mammalian cells. In place of the HA transmembrane domain andintracellular domain, a protease cleavage site is placed at theC-terminal end of the HA extracellular domain, followed by a “foldon”sequence derived from the bacteriophage T4 fibritin to stabilize the HAtrimer, and a C-terminal His-tag to facilitate purification. Therecombinant HA protein is made either as a full-length uncleavedprecursor HA0 with the signal sequence removed or the mature formcontaining HA1 and HA2 subunits with the wild-type fusion peptide at theN-terminus of HA2. In the preferred embodiment, the final purifiedgenetically modified HA forms a trimer as the wild-type HA.

The genes of the designed recombinant influenza HA are made by de novogene synthesis. The gene synthesis technologies have been wellestablished and extensively reviewed (Kosuri, S., and Church, G. M., NatMethods (2014) 11:499-507). Genes of over 10 kb in length have beenroutinely made by commercial providers. Gene synthesis offers theability to modify specific HA sequences and the opportunity ofcodon-optimization according to the expression hosts or specific geneticengineering needs. Restriction sites are designed at specific locationsof the synthesized HA genes to facilitate exchange of HA fragmentsbetween different constructs. The synthesized HA genes with the signalsequences and the protease sites, foldon sequence, and C-terminalHis-tag are incorporated into baculovirus genome with well-establishedprotocols so that the expression of the modified HA proteins is directedby baculovirus polyhedrin promoter. In other embodiments, the same setof HA genes are cloned into mammalian expression vectors for expressionin mammalian cells.

In some embodiments, HA constructs are codon-optimized for expression ininsect cells using baculovirus vectors. In other embodiments, HAconstructs are codon-optimized for expression in mammalian cells,including but not limited to CHO cells (Chinese hamster ovary cells) andHEK 293 cells (human embryonic kidney 293 cells). Codon optimization toany organism is routinely done using commercial software such asLasergene software package from DNASTAR, Inc. (3801 Regent Street,Madison, Wis. 53705 USA), online web server such as OPTIMIZER (locatedon the World Wide Web at genomes.urv.es/OPTIMIZER/), or by genesynthesis service providers using their proprietary algorithms. Codonoptimization takes into considerations of G/C content, elimination ofcryptic splice sites and RNA destabilizing sequence elements, andavoidance of stable RNA secondary structures. Codons are also manuallyadjusted based on the Codon Usage Database (located on the World WideWeb at kazusa.or.jp/codon/). Due to codon degeneracy, gene sequences canbe changed without changing the encoded amino acid sequences.Restriction sites are introduced at specific locations by changingcodons without changing amino acid sequences. Using any of thesemethods, gene sequences, with or without codon optimization to aspecific organism, are routinely generated by back translation ofprotein sequences using a computer algorithm or manually.

Other embodiments include a method to make recombinant non-infectiousinfluenza viruses from the genetically modified HA genes with the MCSchanged to the TEV protease recognition site using established cellculture methods. Reverse-genetics systems that allow production ofinfluenza viruses from RNA segments or plasmids with cloned cDNAs havebeen developed, with or without using helper virus (Luytjes, W., et al.,Cell (1989) 59:1107-1113; Neumann, G., et al., PNAS (1999) 96:9345-9350;Fodor, E., et al., J. Virol (1999) 73:9679-9682; de Wit, E., et al., J.Gen Virol (2007) 88:1281-1287). Infectious influenza viruses are made bytransient transfection of mammalian cells with influenza RNA segments orplasmids with the cDNA of influenza RNAs. These viruses that areisolated from the cell culture media are used to infect embryonatedchicken eggs to produce live infectious influenza viruses for makingvaccines. Some of the influenza RNA segments are replaced by a foreigngene. Recombinant influenza A viruses have been made using an influenzaC virus HEF protein replacing HA protein (Gao, Q., et al., J. Virol(2008) 82:6419-6426). In these embodiments, the cDNA or RNA of themodified HA is co-transfected to mammalian cells with cDNAs of the other7 influenza RNAs or the other 7 RNA segments made by standard moleculartechniques and gene synthesis. Using an established method or similarmethods, multiple identical or different HA proteins can be packaged ina single virion (Uraki, R., et al., J. Virol (2013) 87:7874-7881).

Methods to make recombinant infectious influenza viruses from agenetically modified HA gene use established cell culture methods asdescribed above. In these embodiments, a genetically modified HA genewith H5 HA MCS or polybasic sequence at the MCS is co-transfected tomammalian cells with cDNAs of the other 7 influenza RNAs or the other 7RNA segments made by standard reverse genetics system as describedabove. These modified maturation cleavage sites increase the efficiencyof HA maturation in influenza natural hosts or in cell culture (Kong,W-p., et al., PNAS (2006) 103:15987-15991). The mature functional HAprotein has the ability to bind to host cells and fuse with host cellmembrane.

Other embodiments include a method to make recombinant influenzavirus-like particles (VLP) from the genetically modified HA genes usingestablished cell culture methods in mammalian cells, in insect cells,and in plant cells (Chen, B. J., et al., J. Virol (2007) 81:7111-7123;Smith, G. E., et al., Vaccine (2013) 31:4305-4313; D'Aoust, M. A., etal., Plant Biotech (2010) 8:607-619).

Other embodiments include a method to make DNA vaccines from thegenetically modified HA genes using established methods (Jiang, Y., etal., Antiviral Res (2007) 75:234-241; Alexander, J., et al., Vaccine(2010) 28:664-672; Rao, S. S., et al., PLoS ONE (2010) 5:e9812).

The expression results disclosed herein show that the MCS of H1 HA canbe modified without affecting the expression of the resulting HA.Constructs with H5 HAMCS or polybasic MCS have been demonstrated toexpress to the same level as the wild type H1 HA. Furthermore, the MCSof HA is replaced by TEV protease cleavage site which does not have anybasic residues. By changing the position of the TEV protease cleavagesite, constructs with TEV cleavage site as the MCS have been made thatexpressed to the same level as the wild type H1 HA.

The expression results further show that many of the antigenic sites inthe globular head domain of HA1 can be replaced by heterologous peptidesfrom the stem domain of the same HA or different HAs. Some of theconstructs are expressed to the same level as the wild type HA, whereasother constructs are expressed much less. The nature of the heterologouspeptides and the locations of the heterologous peptides in the globularhead domain have significant impact on the expression level of eachresulting HA construct. More constructs can be made and tested in thesame manner for their expression to identify the most optimal expressionconstructs.

In addition, recombinant HAs are made with replacements of certainimmunodominant antigenic sites of HA globular head domain by M2e epitopefrom influenza M2 protein. Conceivably, any epitope of other influenzaproteins can serve as a replacing peptide to replace an immunodominantantigenic site in the HA globular head domain. Furthermore, aheterologous replacing peptide can be derived from another protein notrelated to influenza viruses. A specific antigenic site in the HAglobular head domain can be chosen for a replacing peptide to maintainthe native structural feature of the replacing peptide as much aspossible to present the replacing peptide to host immune system.

Baculovirus Expression System

Since its introduction about 30 years ago (U.S. Pat. No. 4,745,051;Summers, M. D., and Smith, G. E., (1987) A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures. TexasAgricultural Experiment Station Bulletin No. 1555.), baculovirusexpression vector system (BEVS) has been used to express many differenttypes of human and viral proteins including intracellular proteins,membrane proteins, and secreted proteins. BEVS has been used to producevirus-like particles (VLPs). BEVS is a eukaryotic expression system thatuses insect cells as host, which provides post-translationalmodifications of proteins similar to mammalian cells (Jarvis, D. L.,“Baculovirus Expression Vectors” in The Baculoviruses, ed. Miller, L. K.(1997) pp. 389-420 Plenum Press, New York). BEVS based on Autographacalifornica nuclear polyhedrosis virus (AcNPV) has been wellestablished. Many commercial kits are available for producingrecombinant baculoviruses. BEVS has been successfully used to producerecombinant vaccines marketed in US. Two FDA approved vaccines,Cervarix™, a recombinant human Papillomavirus bivalent (Types 16 and 18)vaccine in the form of non-infectious virus-like particles (VLPs)against cervical cancer (Patent Application WO2010/012780 A1) andFlublok® (U.S. Pat. Nos. 5,762,939 A and 5,858,368 A), a trivalentinfluenza vaccine made of membrane bound hemagglutinin precursors (HA0)against influenza, are produced using BEVS.

To express a target protein using BEVS, the gene-of-interest thatencodes the target protein is first subcloned into a transfer vectorwhich is an E. coli plasmid containing baculovirus sequences flankingthe baculovirus polyhedrin gene. Polyhedrin is an abundant viral proteinin wild type baculoviruses that is not needed for baculoviruspropagation in cell culture. The transfer vector is propagated in E.coli and isolated using standard molecular biology techniques (Sambrook,J., et al., (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor). The transfer vector withgene-of-interest is recombined with baculovirus genomic DNA to produce arecombinant baculovirus genome either in E. coli or in insect cells. Therecombinant baculovirus genome directs the production of the recombinantbaculoviruses and the expression of the target protein. The originalmethod relies on homologous recombination between the same sequencesflanking the polyhedrin gene in transfer vector and in viral genomic DNAin insect cells. The isolated transfer vector DNA is co-transfected withbaculovirus genomic DNA into insect cells. Recombinant baculoviruses areselected for the lack of polyhedrin. Current commercial kits, such asBacPAK™ Baculovirus Expression System from Clontech and BacMagic™ Systemfrom Millipore, use linearized baculovirus genomic DNA that does notproduce viable virus without recombination. These kits allow productionof recombinant baculoviruses with high efficiency and littlecontamination of non-recombinant viruses. Recombination to viral genomecan also be made in vitro using Gateway recombination reaction(BaculoDirect™ Baculovirus Expression System, Life Technologies).Another method to make recombinant viruses is through site-specifictransposition in E. coli (Bac-to-Bac System, Life Technologies). ThepFastBac-based transfer vector contains transposons that make bacmids (alarge plasmid containing the genome of a baculovirus) in E. coli (U.S.Pat. No. 5,348,886). The recombination of the gene-of-interest to abacmid is easily confirmed by polymerase chain reaction (PCR) beforetransfection of bacmids to insect cells.

Recombinant baculoviruses are propagated in insect cell culture. A smallamount of viruses is used to infect insect cells. After a few days, theconditioned media containing amplified viruses are harvested as viralstocks. This amplification process is often repeated several times togenerate a large volume of viral stocks. The viral stocks are routinelystored in the dark with refrigeration for months and even years.Supplement of 5-10% fetal bovine serum (FBS) to the viral stocks hasbeen used to preserve the viral stocks and is believed to prolong theirshelf lives. The viral stocks are sometimes frozen for long term storageat −70° C. Viruses have also been amplified and stored asbaculovirus-infected insect cells (BIIC) (Wasilko, D. J., et al., ProtExp Purif (2009) 65:122-132). The infected insect cells are harvestedbefore their lysis as BIIC stocks and are frozen following standard cellfreezing procedure. BIIC stocks are stored in liquid nitrogen or atultra-low temperature between −65° C. to −85° C. for a long period oftime and are used as viral stocks to infect insect cells for proteinexpression. The frozen BIIC stocks offer longer storage time than viralstocks in liquid form.

The commonly used insect cell lines for BEVS are SF9 and SF21 cellsderived from fall armyworm (Spodoptera frugiperda) and Hi5 or T. nicells derived from cabbage looper (Trichoplusia ni). Other insect cellsderived from species of silkworm (Bombyx mori), honeycomb moth (Galleriamellonella), and gypsy moth (Lymantria dispar) have also been used. SF9,SF21, and Hi5 (or T. ni) cells have been adapted to suspension culturein serum-free media. These cell lines and media are available from manycommercial sources. The cell culture of these cell lines is routinelymaintained in shaker flasks in small volumes of up to 1 or 2 liters inshaker incubators in ambient atmosphere without supplement of gas attemperature in the range of 22-28° C. The cell culture is scaled up instirred tank bioreactors or single-use bioreactors. The conditions forlarge scale insect cell culture in bioreactors have been wellestablished (WAVE Bioreactor Systems—Cell culture procedures. GEHealthcare). Supplement of oxygen is also routinely used in large scaleinsect cell culture to increase cell density.

A series of conditions for expression of a particular target protein aretested to optimize the expression of the target protein using differentcell lines by varying the virus-to-cell ratio and the harvest time postinfection. SF9, SF21, and Hi5 (or T. ni) cells are the most common celllines used for BEVS protein expression. A target protein may expressbetter in one of the cell lines than the others. Some reports show Hi5(or T. ni) cells give more expression of certain secreted proteins. Thevirus-to-cell ratio, commonly known as multiplicity of infection (MOI),is tested to determine the best infection condition for target proteinexpression. Culture samples are taken at various time points postinfection. Cells and the conditioned media are separated bycentrifugation or filtration. Protein expression levels are determinedby standard methods. The culture is monitored for cell density, cellviability, and cell size, which provide the information on cell growthand culture conditions. Glucose level, dissolved oxygen, and pH of theculture are sometimes monitored for consumption of nutrients in themedia. The condition that leads to the best target protein expression isselected for large scale production of the protein.

Although determination of the titers of viral stocks and MOI is widelyused to determine the infection conditions for viral amplification andprotein expression using BEVS, the aforementioned TIPS method offers afaster way to determine the best infection conditions for viralamplification and protein expression. Insect cells enlarge in size afterinfection with recombinant baculoviruses. Cell division also stops afterinfection. Recombinant baculoviruses cause cell lysis around 48 hourspost infection. The cell division is monitored by counting cells as celldensity of the culture. Cell lysis is monitored as cell viability bycounting live and dead cells in the culture. Viable cell size ismeasured as the cell diameter. Cell density, cell viability, and viablecell size are routinely measured by many models of cell countinginstruments. Cell density and cell viability can also be measuredmanually using a hemocytometer. Collectively, cell density, cellviability, and viable cell size provide information on infectionkinetics. TIPS method uses infection kinetics to determine the optimalcondition for expression of a particular target protein using aparticular viral stock. It eliminates the time consuming measurements ofviral titer for MOI calculation. Consistent viral amplification andprotein expression is routinely achieved using TIPS method.

In addition to insect cells, live insects infected with recombinantbaculoviruses have been used to express many secreted proteins andmembrane proteins. Mature HA has been expressed in larvae of tobaccobudworm (Heliothis virescens) (Kuroda, K., et al., J. Virol (1989)63:1677-1685).

Expression system based on other types of baculoviruses such as Bombyxmori nuclear polyhedrosis virus (BmNPV) has also been developed. BmNPVmay have a better biological safety profile over the AcNPV in that ithas a narrower host range and will not grow as insect pests in thefield. BmNPV-based baculovirus expression system has been used toexpress functional proteins in cell culture and silkworm (Bombyx mori)larvae (Maeda, S., et al., Nature (1985) 315:592-594).

BEVS has been routinely used to produce protein complexes and VLPs. Twoor more genes are cloned into a single vector for expression of multipleproteins. Two or more viral stocks each directing the expression of asingle protein are used for co-infection of insect cells to produceprotein complexes or VLPs. Influenza VLPs have been made usingbaculovirus expression system (Bright, R. A., et al., PLoS ONE (2008)3:e1501).

HA Transfer Vectors for Baculovirus Expression

The genes to be expressed using BEVS are commonly under the control ofpolyhedrin promoter, a strong late promoter of baculovirus that leads toprotein expression before the baculovirus-induced insect cell death.Other early or late promoters are also used for protein expression. Tosecrete a recombinant target protein into the cell culture media byinsect cells, a DNA segment that encodes a signal peptide is engineeredat the 5′ end in-frame to the gene-of-interest. Two commonly used signalpeptides for insect cells are the honeybee melittin signal sequence orthe AcNPV envelope surface glycoprotein GP67 signal sequence. SignalSequence Database (located on the World Wide Web atsignalpeptide.de/index.php) lists many other possible signal peptidesfor consideration. After secretion, the signal peptide is removed by acellular protease that processes the signal peptide. Although influenzaviruses are not insect viruses, HA signal peptides have been used assecretion signal peptides for secretion or membrane insertion ofrecombinant proteins in BEVS. A HA signal peptide of MKTIIALSYIFCLVFA iscommonly used for expression of recombinant transmembrane G proteincoupled receptors (GPCRs) in insect cells (Rosenbaum, D. M., et al.,Science (2007) 318:1266-1273; Zou, Y., et al., PLoS One (2012) 7:e46039). HA has been expressed in insect cells using its native signalpeptide. In that particular case, the total expression was lower thanthat using an insect cell signal peptide but the expressed HA was in themature form containing HA1 and HA2 (U.S. Pat. No. 5,858,368 A). The HAsignal peptides are not conserved among HAs. For example, theaforementioned HA signal peptide is different from the H1 HA signalpeptide of SEQ ID NO:7. Empirical selection of HA signal peptides mayimprove HA expression in insect cells.

Commercial vectors are available for making recombinant baculovirusesthrough homologous recombination, using the Bac-to-Bac system or theGateway system (Life Technologies). Features of these transfer vectorssuch as promoters and signal sequences can be customized by standardmolecular biology techniques. The whole transfer vector can now be fullysynthesized by gene synthesis. Gene synthesis allows designs of transfervectors with specific sequences and features.

Methods to express influenza HA using BEVS have been well established atlaboratory scale (Stevens, J., et al., Science (2004) 303:1866-1870).For example, a transfer vector contains the HA from the 1918 influenzavirus under the control of polyhedrin promoter. The 1918 influenza virusHA expression construct has a GP67 signal peptide instead of the HAsignal peptide at the N-terminus for secretion. In place of thetransmembrane domain, a thrombin cleavage site is introduced at theC-terminal end of the HA extracellular domain, followed by a “foldon”sequence derived from the bacteriophage T4 fibritin to stabilize the HAtrimer, and a C-terminal His-tag to facilitate purification. The foldonsequence and His-tag can be removed by thrombin cleavage. Similardesigns of HA expression transfer vectors have been used to expressdifferent HAs from many strains of influenza (Stevens, J., et al.,Science (2006) 312:404-410; Xu, R., et al., Science (2010) 328:357-360;Whittle, J. R. R., et al., PNAS (2011) 108:14216-14221).

In addition to foldon, other trimerization domains can be used tostabilize the trimer of recombinant HAs. For example, a thermostableHIV-1 glycoprotein 41 (gp41) trimerization domain or the initial 16 ofthe 31 residues of the GCN4 leucine zipper sequence was used to aid thetrimerization of truncated HA constructs (Impagliazzo, A., et al.,Science (2015) 349:1301-1306; Yassing, H. M., et al., Nat Med (2015)21:1065-1070).

Recombinant HA precursors (HA0) from both influenza A and influenza Bviruses, with the transmembrane domain and intracellular domain, havebeen made using BEVS at commercial scale as disclosed in U.S. Pat. Nos.5,762,939 A, 5,858,368 A, and 6,245,532 B1. These recombinant HAproteins are the ingredients in Flublok® which has obtained FDA approvalas an influenza vaccine. The HA0 in Flublok® is made using a baculoviruschitinase signal peptide (referred to as 61K signal peptide) to replacethe HA signal peptide. The recombinant HA is associated with theperipheral membrane of insect cells. It is extracted from membrane usingdetergent and further purified.

Mammalian Expression

Chinese hamster ovary (CHO) cells and human embryonic kidney 293(HEK293) cells are commonly used mammalian cell hosts for transienttransfection gene expression of recombinant proteins. Both CHO cells andHEK293 cells have suspension cell lines and adherent cell lines. Thesecells are routinely cultured in media with or without FBS. When cellsare cultured without FBS, chemically defined media are often used. Allsuspension and adherent cell lines can be used to express HA or othersecreted proteins.

Transient transfection is a well-established method to introduce DNAinto cells for protein expression. Many commercial transfection reagentsare available. Similar to the expression optimization process forbaculovirus expression, media samples are taken periodically posttransfection for analysis of protein expression using standard methods.HA proteins are easily detected by affinity capture through the His-tagin the HA constructs or Western blot using many commercially availableanti-HA antibodies. Protein expression usually starts around 48 hourspost transfection and may increase over several days post transfection.Supplements are sometimes added post transfection to increase the targetprotein expression. Other mammalian cell lines such as COS-1, afibroblast-like cell line derived from African green monkey kidneytissue, have also been used for HA expression. Influenza VLPs have beenproduced by co-transfection of multiple plasmids containing cDNAs of all10 influenza virus-encoded proteins (Mena, I., et al., J. Virol (1996)70: 5016-5024; Chen, B. J., et al., J. Virol (2007) 81:7111-7123).

Another method to deliver genes to mammalian cells uses recombinantbaculovirus known as BacMam for baculovirus gene transfer into mammaliancells (Boyce, F. M., and Bucher, N. L., PNAS (1996) 93:2348-2352;Dukkipatia. A., et al., Protein Expr Purif (2008) 62:160-170). Thebaculovirus has been modified by incorporating a mammalian expressioncassette into the baculovirus expression vector for transgene expressionin mammalian cells. Recombinant BacMam baculoviruses are generated bythe standard methods for baculovirus production. Amplified recombinantBacMam baculovirus viral stocks are added to CHO or HEK293 culture fordelivery of the mammalian expression cassette to the CHO or HEK293 cellsfor protein expression. The BacMam platform enables easy transduction oflarge quantities of mammalian cells.

Gene expression vectors transfected to mammalian cells can be integratedinto the cell chromosomes to establish stable cell lines for expressionof the target proteins. Stable CHO cell lines are the most common hostsfor the production of therapeutic biologics such as monoclonalantibodies. The technology is well suited for large scale production oftherapeutic proteins and vaccines. To establish a stable cell line, thecells transfected with an expression vector are subjected to selectionbased on the selection marker on the expression vector. Multiple copiesof the target genes are often integrated into the genome of a stablecell line.

HA Vectors for Mammalian Expression

The HA expression constructs for mammalian expression have the samedesigns and amino acid sequences as those for baculovirus expression.The codons are optimized for CHO cell expression or HEK293 cellexpression. The baculovirus codons also work well for mammalian cellsand vice versa. HA signal peptide has been used as signal peptide forsecretion or membrane insertion of recombinant proteins in mammalianexpression systems. Other commonly used mammalian signal peptidesinclude human IL2 signal peptide, tissue plasminogen activator (tPA)signal peptide, and many other signal peptides found in Signal SequenceDatabase (located on the World Wide Web at signalpeptide.de/index.php).Many mammalian expression vectors are commercially available. Eachvector usually has an enhancer-promoter for high-level expression, apolyadenylation signal and transcription termination sequence for mRNAstability, SV40 origin for episomal replication, antibiotics resistancegene and pUC origin for selection and maintenance in E. coli. Commonlyused promoters for mammalian expression include CMV (Cytomegalovirus)promoter, hEF1-HTLV promoter, a composite promoter comprising theElongation Factor-1α (EF-1α) core promoter and the R segment and part ofthe U5 sequence (R-U5′) of the Human T-Cell Leukemia Virus (HTLV) Type 1Long Terminal Repeat, or other promoters found in MPromDb (MammalianPromoter Database located on the World Wide Web atmpromdb.wistar.upenn.edu/) or The Eukaryotic Promoter Database (EPDlocated on the World Wide Web at epd.vital-it.ch/). A transfer vectoroften has another selection marker for generating stable cell lines.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Construction of Transfer Vectors of H1 HA for RecombinantBaculovirus Production

In this example, a parental plasmid that can be modified to prepare themodified HA protein of the invention is described. The basic outlines ofthis plasmid are shown in FIG. 4 wherein an insert into a standardplasmid for expression in baculovirus is bracketed by restriction sitesfor such insert and restriction sites are present which permitreplacement of the MCS of the HA protein and replacement of theimmunodominant regions in the globular head domain of HA. The encodedprotein is fused to an affinity tag of 10 histidines (10×His-tag) forpurification which can be removed by virtue of an inserted proteasecleavage site after purification. This vector is designated H1WT.

The HA sequence (SEQ ID NO:1) of A/California/07/2009, a swine origininfluenza A virus H1N1 strain that was recommended by WHO for 2009influenza vaccine production (as noted on the World Wide Web atwho.int/csr/resources/publications/swineflu/vaccine_recommendations/en/),was used as the parental sequence to incorporate heterologous peptides.This HA sequence has the accession number of UniProt:C3W5X2. Allconstructs have the same design as illustrated schematically in FIG. 4.The N-terminal HA signal peptide (SEQ ID NO:7) was replaced by GP67signal peptide MVSAIVLYVLLAAAAHSAFA (SEQ ID NO:2). The C-terminaltransmembrane (TM) domain with a sequence of ILAIYSTVASSLVLVVSLGAISFWMCSand intracellular domain with a sequence of NGSLQCRICI of HA wasreplaced by a TEV cleavage site (SEQ ID NO:4) followed by foldon (SEQ IDNO:5) and 10×His-tag (SEQ ID NO:6), namelyGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHH (SEQ ID NO:3), basedon the HA construct first described by Stevens, J., et al., Science(2006) 312:404-410. The foldon stabilizes the recombinant HA while the10×His-tag facilitates purification of HA from cell culture media. Thefoldon and 10×His-tag can be removed by TEV protease that recognizes theTEV cleavage site located between the HA sequence and the foldonsequence. The gene was codon optimized for baculovirus expression andsynthesized by standard method (GENEWIZ, South Plainfield, N.J. 07080,USA).

As shown schematically in FIG. 4, spaced unique restriction sites, ClaIsite, NsiI site, and BamHI site, were introduced into the HA sequence tofacilitate sequence change and swapping. The resulting constructGP67-H1WT-TEV-foldon-10His, denoted as “H1WT” (SEQ ID NO:9), wassubcloned into a pFastBac plasmid between NcoI site and HindIII site.The sequence between ClaI and NsiI sites encodes most of theimmunodominant antigenic sites of the globular head domain. The sequencebetween NsiI and BamHI site encodes the MCS. The expression of H1WTconstruct is under the control of baculovirus polyhedrin promoter. Theresulting plasmid is the transfer vector for the expression of therecombinant wild type HA of A/California/07/2009. The H1WT construct isthe parental construct from which genetically modified HA constructs aremade.

To make genetically modified HA with heterologous peptides, the wildtype fragments of H1WT were replaced using the aforementionedrestriction sites with synthesized DNA fragments that encode specificchanges to the HA. To modify the MCS, the NsiI-BamHI fragment of H1WTwas replaced by DNA fragments that encode altered maturation cleavagesites. To change the antigenic sites in HA1 globular head domain, theClaI-NsiI fragment of H1WT was replaced by DNA fragments that encodealtered antigenic sites with heterologous peptides. Constructs withdifferent combinations of the maturation cleavage sites and theantigenic sites were made by simple exchanges of the restrictionfragments of different plasmids. The resulting constructs were confirmedby DNA sequencing.

Example 2 Design and Construction of H1 HA Constructs with ModifiedMaturation Cleavage Sites

In this example, the segment of H1WT between the NsiI and BamH1restriction sites that bracket the MCS is replaced by alternativesequences that modify this MCS. In several instances, the TEV cleavagesite is included in the modified MCS.

Modified maturation cleavage sites were introduced into the transfervectors of H1 HA described in Example 1. Constructs with modifiedmaturation cleavage sites are listed in Table 1.

TABLE 1 List of H1 HA Constructs with Altered Maturation Cleavage SiteList Construct No. Name Construct Description SEQ ID Number H1MCS^(a)GP67ss-H1MCS-TEV-foldon- 10His^(b) 1 H1WT^(c) GP67ss-H1WT-TEV-foldon-SEQ ID NO: 9 10His 2 H1H5cs GP67ss-H1H5cs-TEV-foldon- SEQ ID NO: 1110His 3 H1R5cs GP67ss-H1R5cs-TEV-foldon- SEQ ID NO: 14 10His 4 H1IR5csGP67ss-H1IR5cs-TEV-foldon- SEQ ID NO: 16 10His 5 H1TEV1GP67ss-H1TEV1-TEV-foldon- SEQ ID NO: 18 10His 6 H1TEV2GP67ss-H1TEV2-TEV-foldon- SEQ ID NO: 20 10His 7 H1TEV3GP67ss-H1TEV3-TEV-foldon- SEQ ID NO: 22 10His ^(a)Constructs name isbased on the maturation cleavage site. H1 denotes H1 HA. MCS denotes amaturation cleavage site. ^(b)Constructs are described by their sequencefeatures. GP67ss denotes GP67 signal sequence. TEV-foldon-10His denotesthe sequence of TEV cleavage site, foldon sequence, and 10xHis-tag atthe C-terminus of the constructs. Schematic drawings of constructs areshown in FIG. 4. ^(c)H1WT is the wild-type H1 HA construct with nativeMCS.

The positions of these modifications are illustrated in FIG. 5.

Construct H1H5cs (SEQ ID NO:11) has the MCS of wild type H1 HA (SEQ IDNO:9) replaced by H5 MCS (SEQ ID NO:12). Construct H1R5cs (SEQ ID NO:14)has the MCS of wild type H1 replaced by five arginines. ConstructH1IR5cs (SEQ ID NO:16) has four arginines inserted between the HA1 andHA2 sequences while keeping all the native HA1 residues. The NsiI-BamHIfragment with the changes were synthesized and subcloned between NsiIand BamHI sites of H1WT to replace the wild type fragment.

To test whether the H1 HA MCS can be modified to the TEV cleavage sitethat does not contain any basic residue, several constructs were made bychanging the number of residues at the H1 HA MCS. Construct H1TEV1 (SEQID NO:18) has 7 residues, PSIQSRG, at the H1 HA MCS replaced by the7-residue TEV cleavage site of ENLYFQG (SEQ ID NO:4). Construct H1TEV2(SEQ ID NO:20) has 8 residues, IPSIQSRG, of the H1 HA MCS replaced bySPENLYFQG containing the TEV cleavage site. Construct H1TEV3 (SEQ IDNO:22) has the last 3 residues (QSR) of H1 HA MCS (SEQ ID NO:8) replacedby the TEV cleavage site.

Example 3 Design and Construction of H1 HA Constructs with HeterologousEpitopes in the Globular Head Domain

In this example, the basic plasmid and the plasmid with modified MCSwere modified between the ClaI and NsiI sites by alternative nucleotidesequences where the various immunodominant sites were replaced withheterologous epitopes.

Heterologous epitopes were introduced to the constructs described inExample 1 and Example 2. The heterologous epitopes were inserted aroundimmunodominant antigenic sites of HA globular head domain or replacedthe immunodominant antigenic sites of HA globular head domain.Constructs with modified antigenic site(s) are listed in Table 2.

TABLE 2 List of H1 HA Constructs with Modified Immunodominant AntigenicSite in the Globular Head Domain List No. Construct Name ConstructDescription SEQ ID No. H1MCS-AS^(a) GP67ss-H1MCS-AS-TEV-foldon-10His^(b)8 H1H5cs-CR8020Ca GP67ss-H1H5cs-CR8020Ca-TEV-foldon-10His SEQ ID NO: 249 H1TEV2-CR8020Ca GP67ss-H1TEV2-CR8020Ca-TEV-foldon-10His SEQ ID NO: 2910 H1TEV2-CR8020Sa3 GP67ss-H1TEV2-CR8020Sa3-TEV- SEQ ID NO: 31foldon-10His 11 H1TEV2-CR8020Sa4 GP67ss-H1TEV2-CR8020Sa4-TEV- SEQ ID NO:33 foldon-10His 12 H1TEV2-I1C9Ca1 GP67ss-H1TEV2-I1C9Ca1-TEV-foldon-10HisSEQ ID NO: 35 13 H1TEV2-I1C9Ca2 GP67ss-H1TEV2-I1C9Ca2-TEV-foldon-10HisSEQ ID NO: 38 14 H1TEV2-I1C9Sa3 GP67ss-H1TEV2-I1C9Sa3-TEV-foldon-10HisSEQ ID NO: 40 15 H1TEV2-I1C9Sa4 GP67ss-H1TEV2-I1C9Sa4-TEV-foldon-10HisSEQ ID NO: 42 16 H1H5cs-I1C9Sb GP67ss-H1H5cs-I1C9Sb-TEV-foldon-10His SEQID NO: 44 17 H1H5cs-I1C9Ca GP67ss-H1H5cs-I1C9Ca-TEV-foldon-10His SEQ IDNO: 46 18 H1H5cs-FI6Sab GP67ss-H1H5cs-FI6Sab-TEV-foldon-10His SEQ ID NO:48 19 H1WT-FI6Sab GP67ss-H1WT-FI6Sab-TEV-foldon-10His SEQ ID NO: 52 20H1TEV1-FI6Sa GP67ss-H1TEV1-FI6Sa-TEV-foldon-10His SEQ ID NO: 54 21H1H5cs-M2eCa GP67ss-H1H5cs-M2eCa2-TEV-foldon-10His SEQ ID NO: 56 22H1H5cs-M2eSb GP67ss-H1H5cs-M2eSb-TEV-foldon-10His SEQ ID NO: 59 23H1TEV2-M2eCa GP67ss-H1TEV2-M2eCa2-TEV-foldon-10His SEQ ID NO: 61^(a)Constructs name is based on the maturation cleavage site andantigenic site modification. H1 denotes H1 HA. MCS denotes a maturationcleavage site. AS denotes an altered immunodominant antigenic site inthe globular head domain of H1 HA with a heterologous epitope. Each ASis indicated by the heterologous epitope and the H1 HA immunodominantantigenic site where the heterologous epitope is placed. ^(b)Constructsare described by their sequence features. GP67ss denotes GP67 signalsequence. TEV-foldon-10His denotes the TEV cleavage site, foldonsequence, and 10xHis-tag at the C-terminus of the constructs. Schematicdrawing of constructs are shown in FIG. 4.

The positions of these modifications are illustrated in FIG. 5.

The composite CR8020 epitope peptide of 13 amino acid residues replaceda surface loop of 12 residues EIAIRPKVRDQE around the antigenic siteCa2. The ClaI-NsiI fragment with composite CR8020 epitope peptidesubstitution was synthesized and subcloned between ClaI and NsiI sitesof H1H5cs to generate a HA construct denoted as H1H5cs-CR8020Ca (SEQ IDNO:24). To make H1TEV2-CR8020Ca (SEQ ID NO:29), the ClaI-NsiI fragmentof H1H5cs-CR8020Ca was isolated and subcloned between ClaI and NsiIsites of H1TEV2.

The construct H1TEV2-CR8020Sa3 (SEQ ID NO:31) has the composite CR8020epitope peptide replacing residues KKGNS of one of the Sa sites, whichcorresponds to the Site B loop of H3 HA on top of the globular headdomain. The ClaI-NsiI fragment with the CR8020 modification wassynthesized and subcloned between ClaI and NsiI sites of H1TEV2 togenerate a HA construct denoted as H1TEV2-CR8020Sa3.

The construct H1TEV2-CR8020Sa4 (SEQ ID NO:33) has the composite CR8020epitope peptide replacing a helical structure of residues KTSS near oneof the Sa sites. This position is on the side of the globular headdomain of HA trimer and away from the receptor binding site. Such amodification is unlikely to change the receptor binding. The ClaI-NsiIfragment with the CR8020 modification was synthesized and subclonedbetween ClaI and NsiI sites of H1TEV2 to generate a HA construct denotedas H1TEV2-CR8020Sa4.

Models of HA monomer of constructs CR8020Sa4 and CR8020Ca are shown inFIG. 6. Each HA monomer has a composite CR8020 epitope peptide in theglobular head domain and the native CR8020 epitope in the stem domain.

Several HA constructs with I1C9 epitope peptide of GIFGAIAGFIEG (SEQ IDNO:36) were made. One construct H1TEV2-I1C9Ca1 (SEQ ID NO:35) has I1C9peptide replacing RPKVRDQE at residues 217 to 224 around Ca2 site.Another construct H1H5cs-I1C9Ca (SEQ ID NO:46) has I1C9 peptidereplacing a longer peptide EIAIRPKVRDQE at residues 213 to 224 of Ca2.Other constructs have I1C9 peptide individually replacing a Ca2 site,HAGAKS, that corresponds to the Site A of H3 HA, an Sa site KKGNS, andan Sa site KTSS. Each of the ClaI-NsiI fragments with the changes wassynthesized and subcloned between ClaI and NsiI sites of H1TEV2 togenerate a HA construct denoted as H1TEV2-I1C9Ca1 (SEQ ID NO:35),H1TEV2-I1C9Ca2 (SEQ ID NO:38), H1TEV2-1C9Sa3 (SEQ ID NO:40), andH1TEV2-I1C9Sa4 (SEQ ID NO:42), respectively. The ClaI-NsiI fragment withthe change of I1C9Ca was synthesized and subcloned between ClaI and NsiIsites of H1H5cs to generate a HA construct H1H5cs-I1C9Ca. The I1C9peptide also replaced residues TSADQQSLYQNA of Sb site of H1H5csconstruct as H1H5cs-I1C9Sb (SEQ ID NO:44) by the same method. The Sbsite corresponds to the Site B helix of H3 HA.

The ClaI-NsiI fragment was gene synthesized with the FI6 epitope peptideRKKRGLFGAIAGFIE (SEQ ID NO:49) placed at the Sa site and the coiled-coilFI6 epitope peptide KESTQKAIDGVTNKVNS (SEQ ID NO:50) placed at the Sbsite. This ClaI-NsiI fragment with FI6 substitutions was subclonedbetween ClaI and NsiI sites of H1H5cs and H1WT, resulting constructsH1H5cs-FI6Sab (SEQ ID NO:48) and H1WT-FI6Sab (SEQ ID NO:52),respectively. Another ClaI-NsiI fragment was gene synthesized with theFI6 epitope peptide RKKRGLFGAIAGFIE (SEQ ID NO:49) placed at the Sasite. This fragment replaced the ClaI-NsiI fragment of H1TEV1 togenerate construct H1TEV1-FI6Sa (SEQ ID NO:54).

Example 4 Design and Construction of H1 HA Constructs with M2e Peptideat Antigenic Sites in Globular Head Domain

In this example, again, the nucleotide sequence of ClaI-NsiI fragmentwas replaced by nucleotide sequences wherein a portion of the Ca2 siteor of the Sb site was replaced by the nucleotide sequence encoding theM2e peptide. The constructs with M2e peptides are listed in Table 2. Thepositions of these modifications are illustrated in FIG. 5.

Gene synthesis generated ClaI-NsiI fragments with M2e peptide (SEQ IDNO:57) replacing EIAIRPKVRDQE at the Ca2 site or replacing helix ofresidues TSADQQSLYQNA of Sb site. HA constructs with M2e peptide werefirst made in H1H5cs, in which the ClaI-NsiI fragment of H1H5cs wasreplaced with individual ClaI-NsiI fragment containing M2e peptide,resulting constructs H1H5cs-M2eCa (SEQ ID NO:56) and H1H5cs-M2eSb (SEQID NO:59), respectively. To transfer the ClaI-NsiI fragment ofH1H5cs-M2eCa to H1TEV2 construct, the ClaI-NsiI fragment of H1H5cs-M2eCawas isolated and subcloned between ClaI and NsiI sites of H1TEV2,resulting a construct named H1TEV2-M2eCa (SEQ ID NO:61).

Example 5 Generation of Recombinant Baculoviruses Using Bac-to-BacBaculovirus Expression Systems

This example describes techniques for preparing insect cells comprisingthe expression systems contained in the constructs prepared in Examples1-4.

Recombinant baculoviruses were generated from the pFastBacbased transfervectors of the constructs following the manufacturer's instruction(Bac-to-Bac Baculovirus Expression Systems, Life Technology, Carlsbad,Calif., USA). Briefly, transfer vectors were transformed to E. coliDH10Bac chemically competent cells. White colonies with recombinantbacmids were selected on plates with Blue-gal. Bacmid DNA was isolatedby standard alkaline lysis miniprep method following the manufacturer'sinstruction. Recombinant bacmids were identified by PCR using the M13Forward (−40) primer (GTTTTCCCAGTCACGAC) and M13 Reverse primer(CAGGAAACAGCTATGAC). Recombinant bacmids gave a PCR product of about 4kb.

SF9 insect cells attached to 12-well plates were transfected with therecombinant bacmids using Cellfectin® Reagent, other commerciallyavailable transfection reagents, or polyethylenimine (PEI). After 4-7days post transfection or when the SF9 cells enlarged due to virusinfection, the cell culture media were harvested as P0 viral stocks.Each P0 viral stock was used to infect 50 ml of SF9 cells at a densityof 2×10⁶ cells/ml in shaker flasks to amplify the viruses. The cultureswere monitored using Cedex Cell Counter (Roche Diagnostics Corporation,Indianapolis, Ind., USA). The harvest time was determined by the averageviable cell size and cell viability. When the average viable cell sizeswere 4-7 μm larger than uninfected cells and viabilities were in therange of 50-70% in 4-5 days post infection, cells were removed bycentrifugation and cell culture supernatants were collected and storedat 4° C. in the dark as P1 viral stocks. Sometimes the viral stocks werefiltered through 0.2 μm sterile filter to maintain sterility.

To further amplify viral stocks, 500 ml of SF9 cells at a density of2×10⁶ cells/ml were infected with 250 μl P1 viral stock. The cultureswere monitored using Cedex Cell Counter. When the average viable cellsizes were 4-7 μm larger than uninfected cells and viabilities were inthe range of 50-70% in 4-5 days post infection, cells were removed bycentrifugation and cell culture supernatants were collected and storedat 4° C. in the dark as P2 viral stocks. Sometimes the viral stocks werefiltered through 0.2 μm sterile filter to maintain sterility.

Example 6 Expression of Recombinant HA Proteins

This example demonstrates expression in insect cells of the variousconstructs described in Examples 1-4. Depending on the specificconstruct, various levels of target protein expression were obtained.

To detect the expression of a recombinant HA, cell culture media of SF9cells that were infected with the recombinant baculovirus stocks,usually the P1 or P2 viral stocks as described in Example 5, werecollected and incubated with Ni-NTA resins (Qiagen, Germantown, Md. USA)for affinity capture through the 10×His-tag of the recombinant HA. Afterwashing the Ni-NTA resin with phosphate buffered saline (1×PBS), theNi-NTA resins were boiled with gel loading buffer in the presence orabsence of a reducing agent for analysis by SDS-PAGE or Western blotusing anti-His antibodies. A protein band around 63 kDa on a Coomassie®stained gel or anti-His Western blot indicates the expression ofsecreted recombinant HA full-length protein.

As shown in FIG. 7 and FIG. 8, the expression of H1 HA protein of theparental construct H1WT was detected as expected. HA proteins withaltered maturation cleavage site of polybasic residues, H1H5cs, H1R5cs,and H1IR5cs, expressed to the same level as H1WT. H1TEV1, with theentire H1 HA maturation cleavage site replaced by the TEV cleavage site,expressed much less than H1WT. By keeping more native residues at thematuration cleavage site, H1TEV2 and H1TEV3 expressed very wellcomparing to H1WT (FIG. 7 and FIG. 8). Not all substitutions expressedto the same level. By adjusting the location of TEV cleavage site in theconstructs, expression level to that of the wild type HA was achieved.

Also shown in FIG. 7 and FIG. 8, placements of the sameimmunosubdominant bNAb epitope at different immunodominant antigenicsites resulted in different expression levels. Constructs of compositeCR8020 epitope peptide at either Sa site (H1TEV2-CR8020Sa4) or at Ca2site (H1H5cs-CR8020Ca) expressed to the same level as the wild type HA,whereas the H1TEV2-CR8020Sa3 construct did not express well. Thus theexact positions of the heterologous epitope placement could be importantfor the expression of the resulting HA. The nature of theimmunosubdominant bNAb epitopes may impact the expression levels of theresulting HA proteins as well.

Individual placement of I1C9 epitope at several antigenic sites alsoshowed different levels of expression of the resulting HA proteins (FIG.7 and FIG. 8). Construct H1H5cs-I1C9Ca showed higher expression thanconstruct H1TEV2-I1C9Ca1. The peptide sequences in the antigenic sitesreplaced by I1C9 epitope peptide in these two constructs are slightlydifferent. Similar to those constructs with TEV cleavage site as MCS,the location of substitution affects the expression level.

Constructs with M2e peptide at Ca2 sites, namely H1H5cs-M2eCa andH1TEV2-M2eCa, gave expression as good as H1WT, regardless of thematuration cleavage sites (FIG. 7 and FIG. 8). However the constructH1H5cs-M2eSb with the M2e peptide at the Sb site did not express well.The Sb site is in a helical structure which may be required for properfolding of HA.

The successful expression of many of the H1 HA constructs demonstratedthat the immunodominant antigenic sites of HA globular head domain canbe replaced by immunosubdominant bNAb epitopes from the stem domain orby an M2e peptide from anther protein not related to HA. These resultsrevealed the plasticity of the globular head domain and validated thefeasibility of making a functional HA to present heterologous epitopesin its globular head domain. Together, Examples 1-6 demonstrated methodsto construct HAs with heterologous epitopes and/or altered MCS and toselect the modified HAs with good expression.

Example 7 Purification of Recombinant HA Proteins

This example demonstrates the purification of the various constructsdescribed in Examples 1-4.

In shaker flasks, 50 ml to 1 L of SF9 cells at a density of 2×10⁶cells/ml were infected with a baculovirus stock at a volumetric ratio of1-4 ml P1 or P2 viral stock to 1 liter of cells. The culture wasmonitored using Cedex Cell Counter. The harvest time was determined bythe average viable cell size and cell viability. When the average viablecell sizes were 4-7 μm larger than uninfected cells and viabilities wereabout 80% in 2 to 3 days post infection, cells were removed bycentrifugation and conditioned cell culture media were collected andstored at 4° C. The conditioned cell culture media were incubated withNi-NTA resins with rocking on a rotisserie platform for 2-4 hours at 4°C. The Ni-NTA resins were collected by centrifugation and washed with1×PBS. The washed Ni-NTA resins were packed into a gravity flow columnand further washed with 1×PBS supplemented with 50 mM imidazole. TheNi-NTA resins were then eluted with 400 mM imidazole in 1×PBS by gravityflow. The eluted HA proteins were buffer exchanged to 1×PBS andconcentrated by ultrafiltration. The purified HA proteins were analyzedby SDS-PAGE as shown in FIG. 8.

APPENDIX

Description of Influenza Viruses, Types and Hosts

Influenza viruses consist of three genera of Orthomyxoviridae family ofviruses, which are influenzavirus A, B and C. Each genus has only asingle species of viruses, namely influenza A, B, and C virus,respectively. Influenza A, B, and C viruses are also called type A, B,and C influenza viruses, respectively. Influenza viruses are envelopednegative-sense single stranded RNA viruses. The viral genome is encodedin separate RNA segments. Influenza A and B viruses each has 8 RNAsegments that encode 10 proteins. Influenza C viruses each has 7 RNAsegments that encode 9 proteins. The 10 characterized proteins ofinfluenza A viruses are PB2, PB1, and PA polymerases, hemagglutinin(HA), nucleoprotein (NP), neuraminidase (NA), matrix proteins M1 and M2,nonstructural proteins NS1 and NS2 (Webster, R. G., et al., MicrobiolRev (1992) 56:152-179). Influenza C viruses have ahemagglutinin-esterase-fusion (HEF) protein that combines the functionsof HA and NA (Herrler, G., et al., J. Gen Virol (1988) 69:839-846).Classification of Influenza A, B and C viruses is based on antigenicdifferences in their NP and matrix proteins.

Influenza A viruses are small particles of 80-120 nm in diameter thatconsist of a host-derived lipid bilayer envelope studded with thevirus-encoded membrane proteins HA, NA, and M2, an inner shell made ofM1 matrix protein, and the nucleocapsids of the viral genome ofindividual RNA segments at the center (Webster, R. G., et al., MicrobiolRev (1992) 56:152-179). The RNA segments are loosely encapsidated bymultiple NP molecules. Complexes of the three viral polymerase proteins(PB1, PB2 and PA) are situated at the ends of the nucleocapsids. All RNAsegments are necessary for producing an infectious virus particle.

RNA segment 1, the slowest-migrating RNA species by gel electrophoresis,encodes PB2 RNA polymerase. RNA segment 2 encodes PB1 RNA polymerase,plus two other transcripts for PB1-N40 and PB1-F2 proteins by usingdifferent reading frames from the same RNA sequence. PB1-N40 and PB1-F2proteins induce host cell apoptosis. RNA segment 3 encodes PA RNApolymerase that forms the RNA-dependent RNA polymerase complex with PB1and PB2. RNA segment 4 encodes HA, the major surface antigen of theinfluenza virion. Each virion has about 500 HA molecules that appears asuniformly distributed spikes on the virion surface (Ruigrok, R. W. H.,et al., J. Gen Virol (1984) 65:799-902; Murti, K. G., and Webster, R.G., Virol (1986) 149:36-43). Binding to the host receptors, HAdetermines the host range. RNA segment 5 encodes NP nucleoprotein. NPencapsidates viral RNA and is transported to host cell nucleus. NP isabundantly synthesized in infected cells and is the second most abundantprotein in the influenza virus virion. It is a major target of the hostcytotoxic T-cell immune response. RNA segment 6 encodes neuraminidase(NA), the second major surface antigen of the influenza virus virion. NAis an enzyme that cleaves terminal sialic acid from glycoproteins orglycolipids to release virus particles from host cell receptors. Itsfunction is required for virus spread. Each virion has about 100 NAmolecules on its surface. NA forms a tetramer and is located in discretepatches on the virion envelope (Murti, K. G., and Webster, R. G., Virol(1986) 149:36-43). RNA segment 7 is bicistronic and encodes both matrixproteins, M1 and M2. M1 forms a shell surrounding the virionnucleocapsids underneath the virion envelope and is the most abundantprotein in the influenza virus virion. M2 is made from a differentsplicing form of the same transcript of M1. M2 is an integral membraneprotein and functions as a proton channel to control the pH of the Golginetwork during the production of influenza virus in host cells. M2 canbe partially replaced by an alternative splicing variant M42. About 3000matrix protein molecules are needed to make one virion. RNA segment 8encodes the two nonstructural proteins NS1 and NS2 for virusreplication. NS2 is from a different reading frame from NS1. Bothproteins are abundant in the infected cell but are not incorporated intoprogeny virions.

Among the three genera of influenza viruses, influenza A viruses are themost virulent human pathogens and cause the most severe disease inhuman. Influenza A viruses are classified into subtypes based on theantigenic properties of their surface glycoproteins HA and NA. A totalof 18 HA subtypes, named H1 to H18, and 10 NA subtypes, named N1 to N10,have been identified (Tong, S., et al., PLoS Pathogens (2013)9:e1003657). The common nomenclature for influenza A subtypes is derivedfrom different combinations of HA subtype and NA subtype, such as H1N1,H3N2, and H7N9 (Bull World Health Organ (1980) 58: 585-591). Waterfowlis the natural reservoir of subtypes H1 to H16 and N1 to N9. SubtypesH17N10 and H18N10 were recently discovered in American fruit bats. Innature, the segmented genome of influenza A viruses allows re-assortmentof RNA segments when a host is co-infected with two subtypes ofinfluenza A viruses. RNA segments from two different subtypes can bepackaged in a single virion to produce a new subtype. Of these manysubtypes of influenza A viruses, only H1N1, H2N2, and H3N2 havedeveloped the capacity for efficient transmission in humans. These threesubtypes are the prevalent virus subtypes of the so called seasonal fluviruses. Human population is therefore immunologically naïve to manyinfluenza A subtypes. Occasionally other subtypes influenza A virusescross species and infect humans to cause pandemic outbreaks with highfatality.

Based on their phylogenetic relationship, influenza A HA subtypes areclustered into two distinct groups. Group 1 includes 10 of the 16subtypes: H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. Group 2accounts for the remaining 6 subtypes: H3, H4, H7, H10, H14, and H15.

Influenza B viruses are antigenically distinct from influenza A viruses.Influenza B viruses co-circulate with type A viruses and cause epidemicsin human. Influenza B viruses are stably adapted to humans without aknown animal reservoir. Unlike the vast genetic variations of HA ofinfluenza A viruses, only one serotype of HA is reported in influenza Bviruses, although three lineages are defined by phylogeneticrelationships of the HA genes. Influenza B viruses do not appear torecombine with influenza A viruses as there has been no report ofre-assortment of RNA segments between influenza A and influenza Bviruses.

Both influenza A and B viruses use terminal sialic acid on host cellsurface as the cellular receptor. HAs of both types of viruses share thesame structural features and bind to the sialic acid receptor for entryto host cells.

Subtypes of influenza A and influenza B viruses are further classifiedinto strains. There are many different strains of influenza A andinfluenza B viruses. Each flu season is dominated by a few strains ofinfluenza A and B viruses that usually differ genetically from thestrains of the previous flu season. Viruses of one strain isolated atdifferent geographic locations or time of a flu season, known asisolates, often have genetic changes.

Infection of influenza C viruses is generally asymptomatic or causesmild illness involving mostly children or young adults. Influenza Cviruses are associated with sporadic cases and minor localizedoutbreaks. Influenza C viruses pose much less of a disease burden thaninfluenza A and B viruses. Large proportion of human population showsseroconversion, which suggests wide circulation of influenza C virusesin human population. Influenza C viruses have also been isolated fromanimals. The hemagglutinin-esterase-fusion (HEF) protein of influenza Cvirus combines the function of HA and NA of the influenza A and Bviruses. Unlike HA of influenza A and B viruses, HEF of influenza Cvirus uses terminal 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) asthe cellular receptor (Herrler, G., et al., EMBO J. (1985) 4:1503-1506).

Description of Virus Infection Process and Life Cycle

Influenza viruses are spread from person to person through respiratorydroplets or fomites (any object or substance capable of carryinginfectious organisms). The viruses infect the epithelial cells of therespiratory tract. After binding to cell surface receptors, the attachedvirion is endocytosed by the host cell to the endosomes where the low pHtriggers a conformational change of HA that leads to insertion of itshydrophobic fusion peptide into the vesicular membrane of the host celland initiate fusion of the viral and vesicular membranes. Fusionreleases the contents of the virion into the cytoplasm of the infectedcell. The viral nucleocapsids migrate into the host cell nucleus, andtheir associated polymerase complexes begin primary transcription ofmRNA for translation of viral proteins. At the same time, translation ofhost mRNAs is blocked. Newly synthesized viral RNAs are encapsidated andviral structural proteins are synthesized and transported to the hostcell surface, where they integrate into the host cell membrane.Influenza viruses bud from the apical surface of polarized epithelialcells, such as bronchial epithelial cells, into lumen of lungs and aretherefore usually pneumotropic (Roth, M. G., et al., PNAS (1979) 76:6430-6434; Nayak, D. P., et al., Virus Res (2009) 143:147-161).Transmission of influenza virus between pigs and humans has beendemonstrated. After an individual becomes infected, the immune systemdevelops antibodies against the influenza virus. This is the body's mainsource of protection.

SEQUENCE LISTINGSEQ ID NO: 1: Peptide sequence of HA of A/California/07/2009, a swine-origininfluenza A virus H1N1 strain (UniProt:C3W5X2)MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREElDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICISEQ ID NO: 2: Peptide sequence of GP67 secretion signal (GP67ss)MVSAIVLYVLLAAAAHSAFASEQ ID NO: 3: Peptide sequence of TEV cleavage site, foldon, and 10xHis-tagGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 4: Peptide sequence of TEV cleavage site ENLYFQGSEQ ID NO: 5: Peptide sequence of foldon GSGYIPEAPRDGQAYVRKDGEWVLLSTFLSEQ ID NO: 6: Peptide sequence of 10xHis-tag HHHHHHHHHHSEQ ID NO: 7: Peptide sequence of signal peptide of HA of A/California/07/2009(UniProt:C3W5X2) MKAILVVLLYTFATANASEQ ID NO: 8: Peptide sequence of H1 HA maturation cleavage site (MCS)IPSIQSRSEQ ID NO: 9: Peptide sequence of GP67ss-H1WT-TEV-foldon-10His (H1WT)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 10: Nucleotide sequence of GP67ss-H1WT-TEV-foldon-10His (H1WT)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCCCTAGCATCCAGAGCAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 11: Peptide sequence of GP67ss-H1H5cs-TEV-foldon-10His (H1H5cs)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO:12: Peptide sequence of H5 maturation cleavage site QRERRKKRSEQ ID NO: 13: Nucleotide sequence of GP67ss-H1H5cs-TEV-foldon-10His (H1H5cs)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 14: Peptide sequence of GP67ss-H1R5cs-TEV-foldon-10His (H1R5cs)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPRRRRRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 15: Nucleotide sequence of GP67ss-H1R5cs-TEV-foldon-10His (H1R5cs)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCCCTAGGAGACGCAGAAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 16: Peptide sequence of GP67ss-H1IR5cs-TEV-foldon-10His (H1IR5cs)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRRRRRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 17: Nucleotide sequence of GP67ss-H1IR5cs-TEV-foldon-10His (H1IR5cs)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCCCTAGCATCCAGAGCAGGAGACGCAGAAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 18: Peptide sequence of GP67ss-H1TEV1-TEV-foldon-10His (H1TEV1)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 19: Nucleotide sequence of GP67ss-H1TEV1-TEV-foldon-10His (H1TEV1)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 20: Peptide sequence of GP67ss-H1TEV2-TEV-foldon-10His (H1TEV2)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 21: Nucleotide sequence of GP67ss-H1TEV2-TEV-foldon-10His (H1TEV2)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 22: Peptide sequence of GP67ss-H1TEV3-TEV-foldon-10His (H1TEV3)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 23: Nucleotide sequence of GP67ss-H1TEV3-TEV-foldon-10His (H1TEV3)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCCCTAGCATCGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 24: Peptide sequence of GP67ss-H1H5cs-CR8020Ca-TEV-foldon-10His (H1H5cs-CR8020Ca)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEGMIDYEGTGQAAGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 25: Peptide sequence of CR8020 epitope of HA2 residues 15-19EGMIDSEQ ID NO: 26: Peptide sequence of CR8020 epitope of HA2 residues 30-36EGTGQAA SEQ ID NO: 27: Peptide sequence of composite C8020 epitopeEGMIDYEGTGQAASEQ ID NO: 28: Nucleotide sequence of GP67ss-H1H5cs-CR8020Ca-TEV-foldon-10His(H1H5cs-CR8020Ca)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAGGCATGATTGATTACGAAGGCACAGGCCAGGCAGCCGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAAaagcttSEQ ID NO: 29: Peptide sequence of GP67ss-H1TEV2-CR8020Ca-TEV-foldon-10His(H1TEV2-CR8020Ca)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEGMIDYEGTGQAAGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 30: Nucleotide sequence of GP67ss-H1TEV2-CR8020Ca-TEV-foldon-10His(H1TEV2-CR8020Ca)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCcTCTGCAcCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCaCaAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAGGCATGATTGATTACGAAGGCACAGGCCAGGCAGCCGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGtGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 31: Peptide sequence of GP67ss-H1TEV2-CR8020Sa3-TEV-foldon-10His (H1TEV2-CR8020Sa3)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVEGMIDYEGTGQAAYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 32: Nucleotide sequence of GP67ss-H1TEV2-CR80205a3-TEV-foldon-10His(H1TEV2-CR8020Sa3)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGGAAGGCATGATTGATTACGAAGGCACAGGCCAGGCAGCCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAAaagcttSEQ ID NO: 33: Peptide sequence of GP67ss-H1TEV2-CR8020Sa4-TEV-foldon-10His (H1TEV2-CR8020Sa4)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPEGMIDYEGTGQAAWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 34: Nucleotide sequence of GP67ss-H1TEV2-CR80205a4-TEV-foldon-10His(H1TEV2-CR8020Sa4)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTGAAGGCATGATTGATTACGAAGGCACAGGCCAGGCAGCCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 35: Peptide sequence of GP67ss-H1TEV2-I1C9Ca1-TEV-foldon-10His (H1TEV2-I1C9Ca1)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIGIFGAIAGFIEGGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 36: Peptide sequence of I1C9 GIFGAIAGFIEGSEQ ID NO: 37: Nucleotide sequence of GP67ss-H1TEV2-I1C9Ca1-TEV-foldon-10His(H1TEV2-I1C9Ca1)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTGGCATTTTCGGCGCTATCGCCGGCTTCATTGAGGGAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 38: Peptide sequence of GP67ss-H1TEV2-I1C9Ca2-TEV-foldon-10His (H1TEV2-I1C9Ca2)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPGIFGAIAGFIEGFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 39: Nucleotide sequence of GP67ss-H1TEV2-I1C9Ca2-TEV-foldon-10His(H1TEV2-I1C9Ca2)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTGGCATTTTCGGCGCTATCGCCGGCTTCATTGAGGGATTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 40: Peptide sequence of GP67ss-H1TEV2-I1C9Sa3-TEV-foldon-10His(H1TEV2-I1C95a3)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVGIFGAIAGFIEGYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 41: Nucleotide sequence of GP67ss-H1TEV2-I1C9Sa3-TEV-foldon-10His (H1TEV2-I1C95a3)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGGGCATTTTCGGCGCTATCGCCGGCTTCATTGAGGGATACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 42: Peptide sequence of GP67ss-H1TEV2-I1C9Sa4-TEV-foldon-10His(H1TEV2-I1C95a4)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPGIFGAIAGFIEGWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 43: Nucleotide sequence of GP67ss-H1TEV2-I1C9Sa4-TEV-foldon-10His(H1TEV2-I1C95a4)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTGGCATTTTCGGCGCTATCGCCGGCTTCATTGAGGGATGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 44: Peptide sequence of GP67ss-H1H5cs-I1C9Sb-TEV-foldon-10His(H1H5cs-I1C9Sb)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSGIFGAIAGFIEGDAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 45: Nucleotide sequence of GP67ss-H1H5cs-I1C9Sb-TEV-foldon-10His(H1H5cs-I1C9Sb)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCGGCATTTTCGGCGCTATCGCCGGCTTCATtGAGGGAGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 46: Peptide sequence of GP67ss-H1H5cs-I1C9Ca-TEV-foldon-10His(H1H5cs-I1C9Ca)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPGIFGAIAGFIEGGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 47: Nucleotide sequence of GP67ss-H1H5cs-I1C9Ca-TEV-foldon-10His(H1H5cs-I1C9Ca)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGGCATTTTCGGCGCTATCGCCGGCTTCATTGAGGGAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGcTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCtGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 48: Peptide sequence of GP67ss-H1H5cs-FI6Sab-TEV-foldon-10His(H1H5cs-FI6Sab)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVRKKRGLFGAIAGFIEYINDKGKEVLVLWGIHHPSKESTQKAIDGVTNKVNSDAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 49: Peptide sequence of maturation cleavage site of FI6 epitopeRKKRGLFGAIAGFIESEQ ID NO: 50: Peptide sequence of helix coiled-coil peptide of FI6 epitopeKESTQKAIDGVTNKVNSSEQ ID NO: 51: Nucleotide sequence of GP67ss-H1H5cs-FI6Sab-TEV-foldon-10His(H1H5cs-FI6Sab)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAGAAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCAAGGAGTCTACACAGAAGGCCATTGATGGCGTTACAAATAAGGTCAATtCTGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAA aagcttSEQ ID NO: 52: Peptide sequence of GP67ss-H1WT-FI6Sab-TEV-foldon-10His(H1WT-FI6Sab)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVRKKRGLFGAIAGFIEYINDKGKEVLVLWGIHHPSKESTQKAIDGVTNKVNSDAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 53: Nucleotide sequence of GP67ss-H1WT-FI6Sab-TEV-foldon-10His(H1WT-FI6Sab)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAGAAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCAAGGAGTCTACACAGAAGGCCATTGATGGCGTTACAAATAAGGTCAATTCTGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCCCTAGCATCCAGAGCAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 54: Peptide sequence of GP67ss-H1TEV1-FI6Sa-TEV-foldon-10His(H1TEV1-FI6Sa)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVRKKRGLFGAIAGFIEGYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 55: Nucleotide sequence of GP67ss-H1TEV1-FI65a-TEV-foldon-10His(H1TEV1-FI6Sa)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAGAAAGAAGAGAGGCCTGTTCGGCGCTATCGCCGGCTTCATTGAGGGATACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATATCGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagcttSEQ ID NO: 56: Peptide sequence of GP67ss-H1H5cs-M2eCa2-TEV-foldon-10His(H1H5cs-M2eCa)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPSLLTEVETPTRNGWECKCSDSGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 57: Peptide sequence of M2e peptide SLLTEVETPTRNGWECKCSDSSEQ ID NO: 58: Nucleotide sequence of GP67ss-H1H5cs-M2eCa2-TEV-foldon-10His(H1H5cs-M2eCa)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTTCCCTGCTGACCGAGGTGGAGACCCCCACCAGGAACGGCTGGGAGTGCAAGTGCTCCGACTCCGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATA AaagcttSEQ ID NO: 59: Peptide sequence of GP67ss-H1H5cs-M2eSb-TEV-foldon-10His(H1H5cs-M2eSb)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSSLLTEVETPTRNGWECKCSDSDAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPQRERRKKRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 60: Nucleotide sequence of GP67ss-H1H5cs-M2eSb-TEV-foldon-10His(H1H5cs-M2eSb)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCTCCCTGCTGACCGAGGTGGAGACCCCCACCAGGAACGGCTGGGAGTGCAAGTGCTCCGACTCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTGAAATTGCCATTAGACCCAAAGTGAGAGATCAGGAAGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTCAGAGGGAGAGACGCAAGAAGAGAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATA AaagcttSEQ ID NO: 61: Peptide sequence of GP67ss-H1TEV2-M2eCa2-TEV-foldon-10His(H1TEV2-M2eCa)MVSAIVLYVLLAAAAHSAFADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPSLLTEVETPTRNGWECKCSDSGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNSPENLYFQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQGAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHHSEQ ID NO: 62: Nucleotide sequence of GP67ss-H1TEV2-M2eCa2-TEV-foldon-10His(H1TEV2-M2eCa)ccATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGATACACTGTGTATTGGCTACCACGCCAACAATAGCACCGATACCGTGGATACAGTGCTGGAGAAGAATGTGACCGTGACCCACTCTGTGAATCTGCTGGAGGATAAGCACAATGGCAAGCTGTGTAAGCTGAGAGGAGTTGCCCCTCTGCACCTGGGCAAATGTAATATTGCCGGCTGGATTCTGGGAAATCCTGAATGTGAAAGCCTGTCTACAGCCAGCAGCTGGTCTTATATCGTGGAAACCCCTAGCAGCGACAATGGCACCTGTTACCCTGGCGACTTCATCGATTACGAGGAGCTGAGAGAACAGCTGTCTAGCGTGTCCAGCTTCGAGAGATTCGAGATCTTCCCTAAGACAAGCAGCTGGCCTAATCACGATTCTAATAAGGGAGTGACAGCCGCCTGTCCTCATGCCGGAGCCAAGTCCTTTTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCTAAGCTGTCTAAGAGCTACATCAACGACAAGGGCAAAGAAGTGCTGGTGCTGTGGGGAATCCACCACCCTAGCACAAGCGCCGATCAGCAGAGCCTGTACCAGAATGCCGATGCCTATGTGTTTGTGGGCAGCAGCAGATACAGCAAAAAGTTCAAGCCTTCCCTGCTGACCGAGGTGGAGACCCCCACCAGGAACGGCTGGGAGTGCAAGTGCTCCGACTCCGGCAGAATGAATTACTACTGGACCCTGGTGGAACCTGGCGATAAGATCACATTTGAGGCCACCGGAAATCTGGTGGTGCCTAGATATGCATTTGCTATGGAGAGAAATGCTGGCTCTGGCATCATTATCTCTGATACCCCTGTGCACGACTGTAATACCACCTGTCAGACACCTAAGGGCGCCATTAATACCAGCCTGCCCTTCCAGAATATTCACCCTATCACCATCGGCAAGTGTCCTAAGTATGTGAAGAGCACCAAGCTGAGACTGGCTACCGGTCTGAGAAATAGCCCTGAAAACCTGTATTTTCAAGGCCTGTTTGGAGCCATCGCCGGCTTTATTGAGGGAGGATGGACCGGAATGGTGGATGGCTGGTACGGCTATCACCACCAGAATGAGCAGGGATCCGGATATGCCGCCGATCTGAAGTCTACACAGAACGCCATCGACGAGATCACAAACAAGGtGAACAGCGTGATCGAGAAGATGAACACCCAGTTTACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATCGAGAACCTGAACAAGAAAGTGGACGACGGCTTCCTGGATATTTGGACCTACAATGCCGAGCTGCTCGTGCTCCTGGAGAATGAGAGAACCCTGGACTACCACGACAGCAATGTGAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAATGCCAAGGAGATCGGCAACGGCTGCTTTGAGTTCTACCACAAGTGTGACAACACCTGTATGGAGTCTGTGAAGAACGGCACCTACGACTACCCTAAGTATAGCGAGGAGGCCAAGCTGAATAGAGAGGAGATCGACGGCGTGAAACTGGAAAGCACAAGAATCTATCAGGGCGCTGAAAACCTGTATTTTCAGGGCGGTTCTGGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTGGGTCACCATCATCACCACCATCACCATCATCACTGATAAaagctt

The invention claimed is:
 1. A modified influenza virus wherein saidvirus comprises an influenza HA protein wherein the maturation cleavagesite (MCS) thereof has been modified so as not to be cleavable by anyanimal proteases but to remain cleavable by a non-animal protease. 2.The modified influenza virus of claim 1 wherein the MCS has beenmodified to be cleavable by a tobacco etch viral (TEV) protease.
 3. Themodified influenza virus of claim 1 wherein the MCS has been cleaved byan appropriate protease.
 4. The modified influenza virus of claim 2wherein the MCS has been cleaved by a TEV protease.
 5. The modifiedinfluenza virus of claim 3 wherein an immunogenic epitope heterologousto the globular region of the HA protein has been inserted into or hasreplaced at least one immunodominant region in the globular head domainof said HA protein.
 6. The modified influenza virus of claim 5 whereinthe heterologous immunogenic epitope is of a tumor associated antigen oran epitope of a virus other than influenza or an alternate influenzaepitope.
 7. The modified influenza virus of claim 6 wherein theheterologous immunogenic epitope is a conserved region of an HA stemregion.
 8. The modified influenza virus of claim 5 wherein more than oneimmunodominant region of said HA protein has been modified to contain aheterologous epitope.
 9. The modified influenza virus of claim 8 whereinat least some of the heterologous epitopes contained in each of saidmore than one immunodominant regions are different from each other. 10.A flu vaccine comprising the modified influenza virus of claim
 1. 11. Aflu vaccine comprising the modified influenza virus of claim
 3. 12. Aflu vaccine comprising the modified influenza virus of claim
 7. 13. Amethod to generate antibodies against flu which method comprisesadministering to a subject the flu vaccine of any of claims 10-12.
 14. Amethod for prophylactic treatment of a subject against flu which methodcomprises administering the vaccine of any of claims 10-12 to saidsubject.
 15. A method to generate antibodies against a desired antigenwhich method comprises administering to a subject the modified influenzavirus of claim
 5. 16. A nucleic acid encoding at least an effectiveportion of an influenza HA protein wherein the nucleotide sequenceencoding the MCS thereof is modified to encode an MCS that is notcleavable by any animal proteases but is cleavable by a non-animalprotease and has not been cleaved.
 17. The nucleic acid of claim 16wherein said MCS is modified to encode an MCS that is cleavable by a TEVprotease.
 18. The nucleic acid of claim 17 wherein an immunogenicepitope heterologous to the globular region of the HA protein has beeninserted into or has replaced at least one immunodominant region in theglobular head domain of said HA protein.
 19. Recombinant host cells thatproduce a modified virus or virus-like particle that comprise the atleast an effective portion of HA protein encoded by the nucleic acid ofclaim
 16. 20. A method to prepare a virus or virus-like particlecomprising a modified HA protein which method comprises culturing therecombinant host cells of claim 19 under conditions wherein said nucleicacid is expressed and recovering the virus or virus-like particlecomprising said at least an effective portion of said HA protein.